Reactions of the Three AmpD Enzymes of <i>Pseudomonas aeruginosa</i>

A group of Gram-negative bacteria, including the problematic pathogen <i>Pseudomonas aeruginosa</i>, has linked the steps in cell-wall recycling with the ability to manifest resistance to β-lactam antibiotics. A key step at the crossroads of the two events is performed by the protease AmpD, which hydrolyzes the peptide in the metabolite that influences these events. In contrast to other organisms that harbor this elaborate system, the genomic sequences of <i>P. aeruginosa</i> reveal it to have three paralogous genes for this protease, designated as <i>ampD</i>, <i>ampDh2</i>, and <i>ampDh3</i>. The recombinant gene products were purified to homogeneity, and their functions were assessed by the use of synthetic samples of three bacterial metabolites in cell-wall recycling and of three surrogates of cell-wall peptidoglycan. The results unequivocally identify AmpD as the <i>bona fide</i> recycling enzyme and AmpDh2 and AmpDh3 as enzymes involved in turnover of the bacterial cell wall itself. These findings define for the first time the events mediated by these three enzymes that lead to turnover of a key cell-wall recycling metabolite as well as the cell wall itself in its maturation

Catalytic Spectrum of the Penicillin-Binding Protein 4 of <i>Pseudomonas aeruginosa</i>, a Nexus for the Induction of β‑Lactam Antibiotic Resistance

<i>Pseudomonas aeruginosa</i> is an opportunistic Gram-negative bacterial pathogen. A primary contributor to its ability to resist β-lactam antibiotics is the expression, following detection of the β-lactam, of the AmpC β-lactamase. As AmpC expression is directly linked to the recycling of the peptidoglycan of the bacterial cell wall, an important question is the identity of the signaling molecule(s) in this relationship. One mechanism used by clinical strains to elevate AmpC expression is loss of function of penicillin-binding protein 4 (PBP4). As the mechanism of the β-lactams is PBP inactivation, this result implies that the loss of the catalytic function of PBP4 ultimately leads to induction of antibiotic resistance. PBP4 is a bifunctional enzyme having both dd-carboxypeptidase and endopeptidase activities. Substrates for both the dd-carboxypeptidase and the 4,3-endopeptidase activities were prepared by multistep synthesis, and their turnover competence with respect to PBP4 was evaluated. The endopeptidase activity is specific to hydrolysis of 4,3-cross-linked peptidoglycan. PBP4 catalyzes both reactions equally well. When <i>P. aeruginosa</i> is grown in the presence of a strong inducer of AmpC, the quantities of both the stem pentapeptide (the substrate for the dd-carboxypeptidase activity) and the 4,3-cross-linked peptidoglycan (the substrate for the 4,3-endopeptidase activity) increase. In the presence of β-lactam antibiotics these altered cell-wall segments enter into the muropeptide recycling pathway, the conduit connecting the sensing event in the periplasm and the unleashing of resistance mechanisms in the cytoplasm

Reactions of All <i>Escherichia coli</i> Lytic Transglycosylases with Bacterial Cell Wall

The reactions of all seven <i>Escherichia coli</i> lytic transglycosylases with purified bacterial sacculus are characterized in a quantitative manner. These reactions, which initiate recycling of the bacterial cell wall, exhibit significant redundancy in the activities of these enzymes along with some complementarity. These discoveries underscore the importance of the functions of these enzymes for recycling of the cell wall

The Natural Product Essramycin and Three of Its Isomers Are Devoid of Antibacterial Activity

Four possible isomers of essramycin, a natural product from a marine <i>Streptomyces</i> species isolated from the Egyptian Mediterranean coast, were synthesized. The structures for the isomers were assigned unequivocally by ¹H NMR, ¹³C NMR, high-resolution mass spectrometry, and X-ray crystal structure determinations. Notwithstanding the earlier report of broad-spectrum antibacterial activity for the natural product, none of the four isomers exhibited any such activity

Turnover of Bacterial Cell Wall by SltB3, a Multidomain Lytic Transglycosylase of <i>Pseudomonas aeruginosa</i>

A family of 11 lytic transglycosylases in <i>Pseudomonas aeruginosa</i>, an opportunistic human pathogen, turn over the polymeric bacterial cell wall in the course of its recycling, repair, and maturation. The functions of these enzymes are not fully understood. We disclose herein that SltB3 of <i>P. aeruginosa</i> is an exolytic lytic transglycosylase. We characterize its reaction and its products by the use of peptidoglycan-based molecules. The enzyme recognizes a minimum of four sugars in its substrate but can process a substrate comprised of a peptidoglycan of 20 sugars. The ultimate product of the reaction is <i>N-</i>acetylglucosamine-1,6-anhydro-<i>N</i>-acetylmuramic acid. The X-ray structure of this enzyme is reported for the first time. The enzyme is comprised of four domains, arranged within an annular conformation. The polymeric linear peptidoglycan substrate threads through the opening of the annulus, as it experiences turnover

Synthesis and Evaluation of 1,2,4-Triazolo[1,5‑<i>a</i>]pyrimidines as Antibacterial Agents Against Enterococcus faecium

Rapid emergence of antibiotic resistance is one of the most challenging global public health concerns. In particular, vancomycin-resistant Enterococcus faecium infections have been increasing in frequency, representing 25% of enterococci infections in intensive care units. A novel class of 1,2,4-triazolo­[1,5-<i>a</i>]­pyrimidines active against E. faecium is reported herein. We used a three-component Biginelli-like heterocyclization reaction for the synthesis of a series of these derivatives based on reactions of aldehydes, β-dicarbonyl compounds, and 3-alkylthio-5-amino-1,2,4-triazoles. The resulting compounds were assayed for antimicrobial activity against the ESKAPE panel of bacteria, followed by investigation of their in vitro activities. These analyses identified a subset of 1,2,4-triazolo­[1,5-<i>a</i>]­pyrimidines that had good narrow-spectrum antibacterial activity against E. faecium and exhibited metabolic stability with low intrinsic clearance. Macromolecular synthesis assays revealed cell-wall biosynthesis as the target of these antibiotics

Structure–Activity Relationship for the 4(3<i>H</i>)‑Quinazolinone Antibacterials

We recently reported on the discovery of a novel antibacterial (<b>2</b>) with a 4­(3<i>H</i>)-quinazolinone core. This discovery was made by in silico screening of 1.2 million compounds for binding to a penicillin-binding protein and the subsequent demonstration of antibacterial activity against <i>Staphylococcus aureus</i>. The first structure–activity relationship for this antibacterial scaffold is explored in this report with evaluation of 77 variants of the structural class. Eleven promising compounds were further evaluated for in vitro toxicity, pharmacokinetics, and efficacy in a mouse peritonitis model of infection, which led to the discovery of compound <b>27</b>. This new quinazolinone has potent activity against methicillin-resistant (MRSA) strains, low clearance, oral bioavailability and shows efficacy in a mouse neutropenic thigh infection model

Cell-Wall Remodeling by the Zinc-Protease AmpDh3 from Pseudomonas aeruginosa

Bacterial cell wall is a polymer of considerable complexity that is in constant equilibrium between synthesis and recycling. AmpDh3 is a periplasmic zinc protease of Pseudomonas aeruginosa, which is intimately involved in cell-wall remodeling. We document the hydrolytic reactions that this enzyme performs on the cell wall. The process removes the peptide stems from the peptidoglycan, the major constituent of the cell wall. We document that the majority of the reactions of this enzyme takes place on the polymeric insoluble portion of the cell wall, as opposed to the fraction that is released from it. We show that AmpDh3 is tetrameric both in crystals and in solution. Based on the X-ray structures of the enzyme in complex with two synthetic cell-wall-based ligands, we present for the first time a model for a multivalent anchoring of AmpDh3 onto the cell wall, which lends itself to its processive remodeling

Reaction Products and the X‑ray Structure of AmpDh2, a Virulence Determinant of Pseudomonas aeruginosa

The zinc protease AmpDh2 is a virulence determinant of Pseudomonas aeruginosa, a problematic human pathogen. The mechanism of how the protease manifests virulence is not known, but it is known that it turns over the bacterial cell wall. The reaction of AmpDh2 with the cell wall was investigated, and nine distinct turnover products were characterized by LC/MS/MS. The enzyme turns over both the cross-linked and noncross-linked cell wall. Three high-resolution X-ray structures, the apo enzyme and two complexes with turnover products, were solved. The X-ray structures show how the dimeric protein interacts with the inner leaflet of the bacterial outer membrane and that the two monomers provide a more expansive surface for recognition of the cell wall. This binding surface can accommodate the 3D solution structure of the cross-linked cell wall

Discovery of Antibiotic (<i>E</i>)‑3-(3-Carboxy­phenyl)-2-(4-cyano­styryl)­quinazolin-4(3<i>H</i>)‑one

In the face of the clinical challenge posed by resistant bacteria, the present needs for novel classes of antibiotics are genuine. <i>In silico</i> docking and screening, followed by chemical synthesis of a library of quinazolinones, led to the discovery of (<i>E</i>)-3-(3-carboxy­phenyl)-2-(4-cyano­styryl)­quinazolin-4­(3<i>H</i>)<i>-</i>one (compound <b>2</b>) as an antibiotic effective <i>in vivo</i> against methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). This antibiotic impairs cell-wall biosynthesis as documented by functional assays, showing binding of <b>2</b> to penicillin-binding protein (PBP) 2a. We document that the antibiotic also inhibits PBP1 of <i>S. aureus</i>, indicating a broad targeting of structurally similar PBPs by this antibiotic. This class of antibiotics holds promise in fighting MRSA infections