11 research outputs found
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 <sup>1</sup>H NMR, <sup>13</sup>C 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