21 research outputs found
Structural and Functional Basis for Targeting <i>Campylobacter jejuni</i> Agmatine Deiminase To Overcome Antibiotic Resistance
<i>Campylobacter jejuni</i> is the most common bacterial
cause of gastroenteritis and a major contributor to infant mortality
in the developing world. The increasing incidence of antibiotic-resistant <i>C. jejuni</i> only adds to the urgency to develop effective
therapies. Because of the essential role that polyamines play, particularly
in protection from oxidative stress, enzymes involved in the biosynthesis
of these metabolites are emerging as promising antibiotic targets.
The recent description of an alternative pathway for polyamine synthesis,
distinct from that in human cells, in <i>C. jejuni</i> suggests
this pathway could be a target for novel therapies. To that end, we
determined X-ray crystal structures of <i>C. jejuni</i> agmatine
deiminase (CjADI) and demonstrated that loss of CjADI function contributes
to antibiotic sensitivity, likely because of polyamine starvation.
The structures provide details of key molecular features of the active
site of this protein. Comparison of the unliganded structure (2.1
Å resolution) to that of the CjADI–agmatine complex (2.5
Ã…) reveals significant structural rearrangements that occur upon
substrate binding. The shift of two helical regions of the protein
and a large conformational change in a loop near the active site generate
a narrow binding pocket around the bound substrate. This change optimally
positions the substrate for catalysis. In addition, kinetic analysis
of this enzyme demonstrates that CjADI is an iminohydrolase that effectively
deiminates agmatine. Our data suggest that <i>C. jejuni</i> agmatine deiminase is a potentially important target for combatting
antibiotic resistance, and these results provide a valuable framework
for guiding future drug development
Acoustofluidic Chemical Waveform Generator and Switch
Eliciting
a cellular response to a changing chemical microenvironment
is central to many biological processes including gene expression,
cell migration, differentiation, apoptosis, and intercellular signaling.
The nature and scope of the response is highly dependent upon the
spatiotemporal characteristics of the stimulus. To date, studies that
investigate this phenomenon have been limited to digital (or step)
chemical stimulation with little control over the temporal counterparts.
Here, we demonstrate an acoustofluidic (i.e., fusion of acoustics
and microfluidics) approach for generating programmable chemical waveforms
that permits continuous modulation of the signal characteristics including
the amplitude (i.e., sample concentration), shape, frequency, and
duty cycle, with frequencies reaching up to 30 Hz. Furthermore, we
show fast switching between multiple distinct stimuli, wherein the
waveform of each stimulus is independently controlled. Using our device,
we characterized the frequency-dependent activation and internalization
of the β<sub>2</sub>-adrenergic receptor (β<sub>2</sub>-AR), a prototypic G-protein coupled receptor (GPCR), using epinephrine.
The acoustofluidic-based programmable chemical waveform generation
and switching method presented herein is expected to be a powerful
tool for the investigation and characterization of the kinetics and
other dynamic properties of many biological and biochemical processes
Acoustofluidic Chemical Waveform Generator and Switch
Eliciting
a cellular response to a changing chemical microenvironment
is central to many biological processes including gene expression,
cell migration, differentiation, apoptosis, and intercellular signaling.
The nature and scope of the response is highly dependent upon the
spatiotemporal characteristics of the stimulus. To date, studies that
investigate this phenomenon have been limited to digital (or step)
chemical stimulation with little control over the temporal counterparts.
Here, we demonstrate an acoustofluidic (i.e., fusion of acoustics
and microfluidics) approach for generating programmable chemical waveforms
that permits continuous modulation of the signal characteristics including
the amplitude (i.e., sample concentration), shape, frequency, and
duty cycle, with frequencies reaching up to 30 Hz. Furthermore, we
show fast switching between multiple distinct stimuli, wherein the
waveform of each stimulus is independently controlled. Using our device,
we characterized the frequency-dependent activation and internalization
of the β<sub>2</sub>-adrenergic receptor (β<sub>2</sub>-AR), a prototypic G-protein coupled receptor (GPCR), using epinephrine.
The acoustofluidic-based programmable chemical waveform generation
and switching method presented herein is expected to be a powerful
tool for the investigation and characterization of the kinetics and
other dynamic properties of many biological and biochemical processes
Acoustofluidic Chemical Waveform Generator and Switch
Eliciting
a cellular response to a changing chemical microenvironment
is central to many biological processes including gene expression,
cell migration, differentiation, apoptosis, and intercellular signaling.
The nature and scope of the response is highly dependent upon the
spatiotemporal characteristics of the stimulus. To date, studies that
investigate this phenomenon have been limited to digital (or step)
chemical stimulation with little control over the temporal counterparts.
Here, we demonstrate an acoustofluidic (i.e., fusion of acoustics
and microfluidics) approach for generating programmable chemical waveforms
that permits continuous modulation of the signal characteristics including
the amplitude (i.e., sample concentration), shape, frequency, and
duty cycle, with frequencies reaching up to 30 Hz. Furthermore, we
show fast switching between multiple distinct stimuli, wherein the
waveform of each stimulus is independently controlled. Using our device,
we characterized the frequency-dependent activation and internalization
of the β<sub>2</sub>-adrenergic receptor (β<sub>2</sub>-AR), a prototypic G-protein coupled receptor (GPCR), using epinephrine.
The acoustofluidic-based programmable chemical waveform generation
and switching method presented herein is expected to be a powerful
tool for the investigation and characterization of the kinetics and
other dynamic properties of many biological and biochemical processes
Acoustofluidic Chemical Waveform Generator and Switch
Eliciting
a cellular response to a changing chemical microenvironment
is central to many biological processes including gene expression,
cell migration, differentiation, apoptosis, and intercellular signaling.
The nature and scope of the response is highly dependent upon the
spatiotemporal characteristics of the stimulus. To date, studies that
investigate this phenomenon have been limited to digital (or step)
chemical stimulation with little control over the temporal counterparts.
Here, we demonstrate an acoustofluidic (i.e., fusion of acoustics
and microfluidics) approach for generating programmable chemical waveforms
that permits continuous modulation of the signal characteristics including
the amplitude (i.e., sample concentration), shape, frequency, and
duty cycle, with frequencies reaching up to 30 Hz. Furthermore, we
show fast switching between multiple distinct stimuli, wherein the
waveform of each stimulus is independently controlled. Using our device,
we characterized the frequency-dependent activation and internalization
of the β<sub>2</sub>-adrenergic receptor (β<sub>2</sub>-AR), a prototypic G-protein coupled receptor (GPCR), using epinephrine.
The acoustofluidic-based programmable chemical waveform generation
and switching method presented herein is expected to be a powerful
tool for the investigation and characterization of the kinetics and
other dynamic properties of many biological and biochemical processes
Acoustofluidic Chemical Waveform Generator and Switch
Eliciting
a cellular response to a changing chemical microenvironment
is central to many biological processes including gene expression,
cell migration, differentiation, apoptosis, and intercellular signaling.
The nature and scope of the response is highly dependent upon the
spatiotemporal characteristics of the stimulus. To date, studies that
investigate this phenomenon have been limited to digital (or step)
chemical stimulation with little control over the temporal counterparts.
Here, we demonstrate an acoustofluidic (i.e., fusion of acoustics
and microfluidics) approach for generating programmable chemical waveforms
that permits continuous modulation of the signal characteristics including
the amplitude (i.e., sample concentration), shape, frequency, and
duty cycle, with frequencies reaching up to 30 Hz. Furthermore, we
show fast switching between multiple distinct stimuli, wherein the
waveform of each stimulus is independently controlled. Using our device,
we characterized the frequency-dependent activation and internalization
of the β<sub>2</sub>-adrenergic receptor (β<sub>2</sub>-AR), a prototypic G-protein coupled receptor (GPCR), using epinephrine.
The acoustofluidic-based programmable chemical waveform generation
and switching method presented herein is expected to be a powerful
tool for the investigation and characterization of the kinetics and
other dynamic properties of many biological and biochemical processes
Mechanism of MenE Inhibition by Acyl-Adenylate Analogues and Discovery of Novel Antibacterial Agents
MenE
is an <i>o</i>-succinylbenzoyl-CoA (OSB-CoA) synthetase
in the bacterial menaquinone biosynthesis pathway and is a promising
target for the development of novel antibacterial agents. The enzyme
catalyzes CoA ligation via an acyl-adenylate intermediate, and we
have previously reported tight-binding inhibitors of MenE based on
stable acyl-sulfonyladenosine analogues of this intermediate, including
OSB-AMS (<b>1</b>), which has an IC<sub>50</sub> value of ≤25
nM for <i>Escherichia coli</i> MenE. Herein, we show that
OSB-AMS reduces menaquinone levels in <i>Staphylococcus aureus</i>, consistent with its proposed mechanism of action, despite the observation
that the antibacterial activity of OSB-AMS is ∼1000-fold lower
than the IC<sub>50</sub> for enzyme inhibition. To inform the synthesis
of MenE inhibitors with improved antibacterial activity, we have undertaken
a structure–activity relationship (SAR) study stimulated by
the knowledge that OSB-AMS
can adopt two isomeric forms in which the OSB side chain exists either
as an open-chain keto acid or a cyclic lactol. These studies revealed
that negatively charged analogues of the keto acid form bind, while
neutral analogues do not, consistent with the hypothesis that the
negatively
charged keto acid form of OSB-AMS is the active isomer.
X-ray crystallography and site-directed mutagenesis confirm the importance
of a conserved arginine for binding the OSB carboxylate. Although
most lactol isomers tested were inactive, a novel difluoroindanediol
inhibitor (<b>11</b>) with improved antibacterial activity was
discovered, providing a pathway toward the development of optimized
MenE inhibitors in the future
Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice
<div><p>Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in <i>orf75A</i> exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host.</p></div
Intraperitoneal administration of MHV68 reveals a reactivation defect in the absence of ORF75A.
<p>C57BL/6 mice were infected at 1000 PFU by the intraperitoneal route with the indicated viruses. <b>(A)</b> Splenomegaly at 18 dpi with indicated viruses. Each symbol represents an individual mouse. Line indicates geometric mean titer. <b>(B)</b> Frequency of splenocytes harboring genomes 18 dpi. <b>(C)</b> Frequency of splenocytes spontaneously reactivating from latency 18 dpi. <b>(D)</b> Reactivation efficiency of splenocytes 18 dpi. <b>(E)</b> Frequency of PECs harboring genomes 18 dpi. <b>(F)</b> Frequency of PECs spontaneously reactivating from latency 18 dpi. For the limiting dilution analyses, curve fit lines were determined by nonlinear regression analysis. Using Poisson analysis, the intersection of the nonlinear regression curves with the dashed line at 63.2% was used to determine the frequency of cells that were either positive for the viral genome or reactivating virus. Data is generated from 5 independent experiments with 4–5 mice per group for splenocyte data and 2 independent experiments with 4 mice per group for PEC data. ** p ≤ 0.005, *** p ≤ 0.0005, and **** p ≤ 0.00005; for A significance determined by two-way unpaired t-test; for B-F significance determined by two-way paired t-test.</p
Frequencies of cell populations reactivating viral genomes in C57BL/6 mice.
<p>Frequencies of cell populations reactivating viral genomes in C57BL/6 mice.</p