bluuebunny/crossref_metadata_2025_split huggingface dataset:
Safety, Tolerability, and Pharmacokinetics of MEDI4893, an Investigational, Extended-Half-Life, Anti-Staphylococcus aureus Alpha-Toxin Human Monoclonal Antibody, in Healthy Adults
Authors: Yu Xiang-Qing , Robbie Gabriel J. , Wu Yuling , Esser Mark T. , Jensen Kathryn , Schwartz Howard I. , Bellamy Terramika , Hernandez-Illas Martha , Jafri Hasan S. 
Published: October 2016

ABSTRACT

MEDI4893 is an investigational immunoglobulin G1(κ) monoclonal antibody that specifically binds to and neutralizes alpha-toxin, a key
Staphylococcus aureus
virulence factor. A triple-amino-acid substitution, M252Y/S254T/T256E, was engineered into the MEDI4893 Fc region to extend its serum half-life. A phase 1, double-blind, dose escalation study was designed to evaluate the safety, tolerability, pharmacokinetics, anti-alpha-toxin-neutralizing activity, and antidrug antibody (ADA) response of MEDI4893 following a single intravenous infusion in healthy adults 18 to 65 years of age. Thirty-three subjects were randomly assigned to receive MEDI4893 at 225 mg (
n
= 3), 750 mg (
n
= 3), 2,250 mg (
n
= 8), or 5,000 mg (
n
= 12) or placebo (
n
= 7) and were followed for 360 days. Adverse events were mild or moderate in severity; none were serious. The MEDI4893 peak serum concentration increased dose proportionally from 77.2 μg/ml (225-mg dose) to 1,784 μg/ml (5,000-mg dose). The area under the concentration-time curve from 0 to 360 days also increased dose proportionally, from 4,840 μg · day/ml (225-mg dose) to 91,493 μg · day/ml (5,000-mg dose), indicating linear pharmacokinetics. MEDI4893's terminal half-life was estimated to be 80 to 112 days, which is approximately 4-fold longer than the half-lives of other human immunoglobulin G antibodies. The alpha-toxin-neutralizing activity in serum correlated highly with the MEDI4893 concentrations in serum. Three adults transiently tested positive for ADA on day 151, but this did not have an impact on MEDI4893 serum concentrations or the MEDI4893 safety profile; no subjects exhibited serum ADA at the study end. These data support the continued development of MEDI4893 for the prevention of
S. aureus
-mediated pneumonia. (This study has been registered at ClinicalTrials.gov under identifier NCT02296320.)
Preclinical Characterization and Human Microdose Pharmacokinetics of ITMN-8187, a Nonmacrocyclic Inhibitor of the Hepatitis C Virus NS3 Protease
Authors: Rajagopalan Ravi , Pan Lin , Schaefer Caralee , Nicholas John , Lim Sharlene , Misialek Shawn , Stevens Sarah , Hooi Lisa , Aleskovski Natalia , Ruhrmund Donald , Kossen Karl , Huang Lea , Yap Sophia , Beigelman Leonid , Serebryany Vladimir , Liu Jyanwei , Sastry Srikonda , Seiwert Scott , Buckman Brad 
Published: October 2016

Abstract

The current paradigm for the treatment of chronic hepatitis C virus (HCV) infection involves combinations of agents that act directly on steps of the HCV life cycle. Here we report the preclinical characteristics of ITMN-8187, a nonmacrocyclic inhibitor of the NS3/4A HCV protease. X-ray crystallographic studies of ITMN-8187 and simeprevir binding to NS3/4A protease demonstrated good agreement between structures. Low nanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 1, 2b, 4, 5, and 6. In cell-based potency assays, half-maximal reduction of genotype 1a and 1b HCV replicon RNA was afforded by 11 and 4 nM doses of ITMN-8187, respectively. Combinations of ITMN-8187 with other directly acting antiviral agents
in vitro
displayed additive antiviral efficacy. A 30-mg/kg of body weight dose of ITMN-8187 administered for 4 days yielded significant viral load reductions through day 5 in a chimeric mouse model of HCV. A 3-mg/kg oral dose administered to rats, dogs, or monkeys yielded concentrations in plasma 16 h after dosing that exceeded the half-maximal effective concentration of ITMN-8187. Human microdose pharmacokinetics showed low intersubject variability and prolonged oral absorption with first-order elimination kinetics compatible with once-daily dosing. These preclinical characteristics compare favorably with those of other NS3/4A inhibitors approved for the treatment of chronic HCV infection.
The Helicobacter pylori Autotransporter ImaA Tempers the Bacterium's Interaction with α
5
β
1
Integrin
Authors: Sause William E. , Keilberg Daniela , Aboulhouda Soufiane , Ottemann Karen M. 
Published: October 2016

ABSTRACT

The human pathogen
Helicobacter pylori
uses the host receptor α
5
β
1
integrin to trigger inflammation in host cells via its
cag
pathogenicity island (
cag
PAI) type IV secretion system (T4SS). Here, we report that the
H. pylori
ImaA protein (HP0289) decreases the action of the
cag
PAI T4SS via tempering the bacterium's interaction with α
5
β
1
integrin. Previously,
imaA
-null mutants were found to induce an elevated inflammatory response that was dependent on the
cag
PAI T4SS; here we extend those findings to show that the elevated response is independent of the CagA effector protein. To understand how ImaA could be affecting
cag
PAI T4SS activity at the host cell interface, we utilized the Phyre structural threading program and found that ImaA has a region with remote homology to bacterial integrin-binding proteins. This region was required for ImaA function. Unexpectedly, we observed that
imaA
mutants bound higher levels of α
5
β
1
integrin than wild-type
H. pylori
, an outcome that required the predicted integrin-binding homology region of ImaA. Lastly, we report that ImaA directly affected the amount of host cell β1 integrin but not other cellular integrins. Our results thus suggest a model in which
H. pylori
employs ImaA to regulate interactions between integrin and the T4SS and thus alter the host inflammatory strength.
Purine Biosynthesis Metabolically Constrains Intracellular Survival of Uropathogenic Escherichia coli
Authors: Shaffer Carrie L. , Zhang Ellisa W. , Dudley Anne G. , Dixon Beverly R. E. A. , Guckes Kirsten R. , Breland Erin J. , Floyd Kyle A. , Casella Daniel P. , Algood Holly M. Scott , Clayton Douglass B. , Hadjifrangiskou Maria 
Published: October 2016

ABSTRACTThe ability tode novosynthesize purines has been associated with the intracellular survival of multiple bacterial pathogens. UropathogenicEscherichia coli(UPEC), the predominant cause of urinary tract infections, undergoes a transient intracellular lifestyle during which bacteria clonally expand into multicellular bacterial communities within the cytoplasm of bladder epithelial cells. Here, we characterized the contribution of the conservedde novopurine biosynthesis-associated locuscvpA-purFto UPEC pathogenesis. Deletion ofcvpA-purF, or ofpurFalone, abolishedde novopurine biosynthesis but did not impact bacterial adherence propertiesin vitroor in the bladder lumen. However, upon internalization by bladder epithelial cells, UPEC deficient inde novopurine biosynthesis was unable to expand into intracytoplasmic bacterial communities over time, unless it was extrachromosomally complemented. These findings indicate that UPEC is deprived of purine nucleotides within the intracellular niche and relies onde novopurine synthesis to meet this metabolic requirement.
Negative Regulation of Ectoine Uptake and Catabolism in Sinorhizobium meliloti: Characterization of the EhuR Gene
Authors: Yu Qinli , Cai Hanlin , Zhang Yanfeng , He Yongzhi , Chen Lincai , Merritt Justin , Zhang Shan , Dong Zhiyang 
Published: October 2016

ABSTRACT

Ectoine has osmoprotective effects on
Sinorhizobium meliloti
that differ from its effects in other bacteria. Ectoine does not accumulate in
S. meliloti
cells; instead, it is degraded. The products of the
ehuABCD-eutABCDE
operon were previously discovered to be responsible for the uptake and catabolism of ectoine in
S. meliloti
. However, the mechanism by which ectoine is involved in the regulation of the
ehuABCD-eutABCDE
operon remains unclear. The
ehuR
gene, which is upstream of and oriented in the same direction as the
ehuABCD-eutABCDE
operon, encodes a member of the MocR/GntR family of transcriptional regulators. Quantitative reverse transcription-PCR and promoter-
lacZ
reporter fusion experiments revealed that EhuR represses transcription of the
ehuABCD-eutABCDE
operon, but this repression is inhibited in the presence of ectoine. Electrophoretic mobility shift assays and DNase I footprinting assays revealed that EhuR bound specifically to the DNA regions overlapping the −35 region of the
ehuA
promoter and the +1 region of the
ehuR
promoter. Surface plasmon resonance assays further demonstrated direct interactions between EhuR and the two promoters, although EhuR was found to have higher affinity for the
ehuA
promoter than for the
ehuR
promoter.
In vitro
, DNA binding by EhuR could be directly inhibited by a degradation product of ectoine. Our work demonstrates that EhuR is an important negative transcriptional regulator involved in the regulation of ectoine uptake and catabolism and is likely regulated by one or more end products of ectoine catabolism.

IMPORTANCE
Sinorhizobium meliloti
is an important soil bacterium that displays symbiotic interactions with legume hosts. Ectoine serves as a key osmoprotectant for
S. meliloti
. However, ectoine does not accumulate in the cells; rather, it is degraded. In this study, we characterized the transcriptional regulation of the operon responsible for ectoine uptake and catabolism in
S. meliloti
. We identified and characterized the transcription repressor EhuR, which is the first MocR/GntR family member found to be involved in the regulation of compatible solute uptake and catabolism. More importantly, we demonstrated for the first time that an ectoine catabolic end product could modulate EhuR DNA-binding activity. Therefore, this work provides new insights into the unique mechanism of ectoine-induced osmoprotection in
S. meliloti
.
Cell-Biological Studies of Osmotic Shock Response in Streptomyces spp
Authors: Fuchino Katsuya , Flärdh Klas , Dyson Paul , Ausmees Nora 
Published: October 2016

ABSTRACTMost bacteria are likely to face osmotic challenges, but there is yet much to learn about how such environmental changes affect the architecture of bacterial cells. Here, we report a cell-biological study in model organisms of the genusStreptomyces, which are actinobacteria that grow in a highly polarized fashion to form branching hyphae. The characteristic apical growth ofStreptomyceshyphae is orchestrated by protein assemblies, called polarisomes, which contain coiled-coil proteins DivIVA and Scy, and recruit cell wall synthesis complexes and the stress-bearing cytoskeleton of FilP to the tip regions of the hyphae. We monitored cell growth and cell-architectural changes by time-lapse microscopy in osmotic upshift experiments. Hyperosmotic shock caused arrest of growth, loss of turgor, and hypercondensation of chromosomes. The recovery period was protracted, presumably due to the dehydrated state of the cytoplasm, before hyphae could restore their turgor and start to grow again. In most hyphae, this regrowth did not take place at the original hyphal tips. Instead, cell polarity was reprogrammed, and polarisomes were redistributed to new sites, leading to the emergence of multiple lateral branches from which growth occurred. Factors known to regulate the branching pattern ofStreptomyceshyphae, such as the serine/threonine kinase AfsK and Scy, were not involved in reprogramming of cell polarity, indicating that different mechanisms may act under different environmental conditions to control hyphal branching. Our observations of hyphal morphology during the stress response indicate that turgor and sufficient hydration of cytoplasm are required forStreptomycestip growth.IMPORTANCEPolar growth is an intricate manner of growth for accomplishing a complicated morphology, employed by a wide range of organisms across the kingdoms of life. The tip extension ofStreptomyceshyphae is one of the most pronounced examples of polar growth among bacteria. The expansion of the cell wall by tip extension is thought to be facilitated by the turgor pressure, but it was unknown how external osmotic change influencesStreptomycestip growth. We report here that severe hyperosmotic stress causes cessation of growth, followed by reprogramming of cell polarity and rearrangement of growth zones to promote lateral hyphal branching. This phenomenon may represent a strategy of hyphal organisms to avoid osmotic stress encountered by the growing hyphal tip.
Characterization of a Novel Iron Acquisition Activity That Coordinates the Iron Response with Population Density under Iron-Replete Conditions in Bacillus subtilis
Authors: Roy Emily M. , Griffith Kevin L. 
Published: October 2016

ABSTRACT

Iron is an essential micronutrient required for the viability of many organisms. Under oxidizing conditions, ferric iron is highly insoluble (∼10
−9
to 10
−18
M), yet bacteria typically require ∼10
−6
M for survival. To overcome this disparity, many bacteria have adopted the use of extracellular iron-chelating siderophores coupled with specific iron-siderophore uptake systems. In the case of
Bacillus subtilis
, undomesticated strains produce the siderophore bacillibactin. However, many laboratory strains, e.g., JH642, have lost the ability to produce bacillibactin during the process of domestication. In this work, we identified a novel iron acquisition activity from strain JH642 that accumulates in the growth medium and coordinates the iron response with population density. The molecule(s) responsible for this activity was named
e
lemental
Fe
(II/III) (Efe) acquisition factor because
efeUOB
(
ywbLMN
) is required for its activity. Unlike most iron uptake molecules, including siderophores and iron reductases, Efe acquisition factor is present under iron-replete conditions and is regulated independently of Fur repressor. Restoring bacillibactin production in strain JH642 inhibits the activity of Efe acquisition factor, presumably by sequestering available iron. A similar iron acquisition activity is produced from a mutant of
Escherichia coli
unable to synthesize the siderophore enterobactin. Given the conservation of
efeUOB
and its regulation by catecholic siderophores in
B. subtilis
and
E. coli
, we speculate that Efe acquisition factor is utilized by many bacteria, serves as an alternative to Fur-mediated iron acquisition systems, and provides cells with biologically available iron that would normally be inaccessible during aerobic growth under iron-replete conditions.

IMPORTANCE
Iron is an essential micronutrient required for a variety of biological processes, yet ferric iron is highly insoluble during aerobic growth. In this work, we identified a novel iron acquisition activity that coordinates the iron response with population density in laboratory strains of
Bacillus subtilis
. We named the molecule(s) responsible for this activity elemental Fe(II/III) (Efe) acquisition factor after the
efeUOB
(
ywbLMN
) operon required for its uptake into cells. Unlike most iron uptake systems, Efe acquisition factor is present under iron-replete conditions and is regulated independently of Fur, the master regulator of the iron response. We speculate that Efe acquisition factor is highly conserved among bacteria and serves as a backup to Fur-mediated iron acquisition systems.
EssD, a Nuclease Effector of the Staphylococcus aureus ESS Pathway
Authors: Ohr Ryan Jay , Anderson Mark , Shi Miaomiao , Schneewind Olaf , Missiakas Dominique 
Published: October 2016

ABSTRACT

Specialized secretion systems of bacteria evolved for selective advantage, either killing microbial competitors or implementing effector functions during parasitism. Earlier work characterized the
E
SAT-6
s
ecretion
s
ystem (ESS) of
Staphylococcus aureus
and demonstrated its contribution to persistent staphylococcal infection of vertebrate hosts. Here, we identify a novel secreted effector of the ESS pathway, EssD, that functions as a nuclease and cleaves DNA but not RNA. EssI, a protein of the DUF600 family, binds EssD to block its nuclease activity in the staphylococcal cytoplasm. An
essD
knockout mutant or a variant lacking nuclease activity,
essD
L546P
, elicited a diminished interleukin-12 (IL-12) cytokine response following bloodstream infection of mice, suggesting that the effector function of EssD stimulates immune signaling to support the pathogenesis of
S. aureus
infections.

IMPORTANCE
Bacterial type VII or ESAT-6-like secretion systems (ESS) may have evolved to modulate host immune responses during infection, thereby contributing to the pathogenesis of important diseases such as tuberculosis and methicillin-resistant
S. aureus
(MRSA) infection. The molecular mechanisms whereby type VII secretion systems achieve their goals are not fully elucidated as secreted effectors with biochemical functions have heretofore not been identified. We show here that MRSA infection relies on the secretion of a nuclease effector that cleaves DNA and contributes to the stimulation of IL-12 signaling during infection. These results identify a biological mechanism for the contribution of the ESS pathway toward the establishment of MRSA disease.
Role of O
2
in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate
Authors: Wheatley Rachel M. , Ramachandran Vinoy K. , Geddes Barney A. , Perry Benjamin J. , Yost Chris K. , Poole Philip S. 
Published: October 2016

ABSTRACT

Insertion sequencing (INSeq) analysis of
Rhizobium leguminosarum
bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O
2
was used to understand how O
2
concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 [
eryD
] and RL0547 [
phoB
]), five were required on succinate (pRL100388, RL1641, RL1642, RL3427, and RL4524 [
ecfL
]), and three were required on 1% O
2
(pRL110072, RL0545 [
phoU
], and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmidless rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the Entner-Doudoroff (ED) pathway and tricarboxylic acid (TCA) cycle for optimal growth on glucose. Surprisingly, the ED pathway was required for growth on succinate, suggesting that sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 (
gatB
) and pRL120419 (
opaA
, encoding omega-amino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis, including ribose-phosphate pyrophosphokinase (RL3468 [
prs
]) and a cytosine deaminase (pRL90208 [
codA
]). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 (
glnB
, encoding nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O
2
, conditions similar to those experienced by N
2
-fixing bacteroids. Glutamate synthesis is constitutively activated in
glnB
mutants, suggesting that consumption of 2-ketoglutarate may increase flux through the TCA cycle, leading to excess reductant that cannot be reoxidized at 1% O
2
and cell death.

IMPORTANCE
Rhizobium leguminosarum
, a soil bacterium that forms N
2
-fixing symbioses with several agriculturally important leguminous plants (including pea, vetch, and lentil), has been widely utilized as a model to study
Rhizobium
-legume symbioses. Insertion sequencing (INSeq) has been used to identify factors needed for its growth on different carbon sources and O
2
levels. Identification of these factors is fundamental to a better understanding of the cell physiology and core metabolism of this bacterium, which adapts to a variety of different carbon sources and O
2
tensions during growth in soil and N
2
fixation in symbiosis with legumes.
pqiABC
and
yebST
, Putative
mce
Operons of Escherichia coli, Encode Transport Pathways and Contribute to Membrane Integrity
Authors: Nakayama Takayuki , Zhang-Akiyama Qiu-Mei 
Published: October 2016

ABSTRACT

The membranes of single-cell organisms are crucial as the first line of defense. The outer membrane of Gram-negative bacteria is an asymmetric bilayer in which lipopolysaccharides (LPSs) and phospholipids are localized in the outer and inner leaflet, respectively. This asymmetry is important for membrane integrity. In
Escherichia coli
, the Mla transport pathway maintains this asymmetry by removing phospholipids from the outer leaflet. The MlaD component of this system is a mammalian cell entry (MCE) domain protein, and
E. coli
has two other MCE domain proteins of unknown function (PqiB and YebT). Here, we show that these two proteins are components of novel transport pathways that contribute to membrane integrity. The
pqiAB
operon is regulated by SoxS and RpoS. The
yebST
operon contains
pqiAB
homologues. Here, we found a third member of the
pqi
operon,
ymbA
(
pqiC
). A PqiB-PqiC complex bridges the inner and the outer membrane, and in other bacteria,
pqiBC
genes are located in operons together with transporter proteins. We show here that simultaneous deletion of
pqiABC
and
yebST
operons in an Δ
mla
background rendered cells more sensitive to SDS-EDTA, and the SDS-EDTA sensitivity of
mla
mutants was rescued by additional copies of
pqiABC
. We also found that the
yebST
operon was induced by a defect in LPS molecules. In conclusion, PqiABC and YebST are novel transport pathways related to the Mla transport pathway and important for membrane integrity.

IMPORTANCE
Membranes of bacteria are crucial for stress resistance. The composition of the
E. coli
outer membrane is asymmetric, with asymmetry maintained by the Mla ABC transport pathway. We propose that the stress-inducible
pqiABC
operon and homologous
yebST
operon, both of previously unknown function, encode transport pathway proteins related to the Mla transport pathway. Deletion of these operons rendered cells more sensitive to membrane stress, and additional copies of
pqiABC
suppressed the SDS-EDTA sensitivity of
mla
mutant strains. We found that
yebS′-′lacZ
fusion was activated in mutant strains with defective LPS molecules.