bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Dosing Strategies Increase Exposures of the Potent Antituberculosis Drug Rifapentine but Are Poorly Tolerated in Healthy Volunteers
Authors: Dooley Kelly E. , Savic Radojka M. , Park Jeong-Gun , Cramer Yoninah , Hafner Richard , Hogg Evelyn , Janik Jennifer , Marzinke Mark A. , Patterson Kristine , Benson Constance A. , Hovind Laura , Dorman Susan E. , Haas David W. ,   ACTG A5311 Study Team
Published: April 2015

ABSTRACT

Rifapentine is a potent antituberculosis drug currently in phase III trials. Bioavailability decreases with increasing dose, yet high daily exposures are likely needed to improve efficacy and shorten the tuberculosis treatment duration. Further, the limits of tolerability are poorly defined. The phase I multicenter trial in healthy adults described here investigated two strategies to increase rifapentine exposures: dividing the dose or giving the drug with a high-fat meal. In arm 1, rifapentine was administered at 10 mg/kg of body weight twice daily and 20 mg/kg once daily, each for 14 days, separated by a 28-day washout; the dosing sequence was randomized. In arm 2, 15 mg/kg rifapentine once daily was given with a high-fat versus a low-fat breakfast. Sampling for pharmacokinetic analysis was performed on days 1 and 14. Population pharmacokinetic analyses were performed. This trial was stopped early for poor tolerability and because of safety concerns. Of 44 subjects, 20 discontinued prematurely; 11 of these discontinued for protocol-defined toxicity (a grade 3 or higher adverse event or grade 2 or higher rifamycin hypersensitivity). Taking rifapentine with a high-fat meal increased the median steady-state area under the concentration-time curve from time zero to 24 h (AUC
0–24ss
) by 31% (relative standard error, 6%) compared to that obtained when the drug was taken with a low-fat breakfast. Dividing the dose increased exposures substantially (e.g., 38% with 1,500 mg/day). AUC
0–24ss
was uniformly higher in our study than in recent tuberculosis treatment trials, in which toxicity was rare. In conclusion, two strategies to increase rifapentine exposures, dividing the dose or giving it with a high-fat breakfast, successfully increased exposures, but toxicity was common in healthy adults. The limits of tolerability in patients with tuberculosis remain to be defined. (AIDS Clinical Trials Group study A5311 has been registered at ClinicalTrials.gov under registration no. NCT01574638.)
Inhibition of Outer Membrane Proteases of the Omptin Family by Aprotinin
Authors: Brannon John R. , Burk David L. , Leclerc Jean-Mathieu , Thomassin Jenny-Lee , Portt Andrea , Berghuis Albert M. , Gruenheid Samantha , Le Moual Hervé 
Published: April 2015

ABSTRACT

Bacterial proteases are important virulence factors that inactivate host defense proteins and contribute to tissue destruction and bacterial dissemination. Outer membrane proteases of the omptin family, exemplified by
Escherichia coli
OmpT, are found in some Gram-negative bacteria. Omptins cleave a variety of substrates at the host-pathogen interface, including plasminogen and antimicrobial peptides. Multiple omptin substrates relevant to infection have been identified; nonetheless, an effective omptin inhibitor remains to be found. Here, we purified native CroP, the OmpT ortholog in the murine pathogen
Citrobacter rodentium
. Purified CroP was found to readily cleave both a synthetic fluorescence resonance energy transfer substrate and the murine cathelicidin-related antimicrobial peptide. In contrast, CroP was found to poorly activate plasminogen into active plasmin. Although classical protease inhibitors were ineffective against CroP activity, we found that the serine protease inhibitor aprotinin displays inhibitory potency in the micromolar range. Aprotinin was shown to act as a competitive inhibitor of CroP activity and to interfere with the cleavage of the murine cathelicidin-related antimicrobial peptide. Importantly, aprotinin was able to inhibit not only CroP but also
Yersinia pestis
Pla and, to a lesser extent,
E. coli
OmpT. We propose a structural model of the aprotinin-omptin complex in which Lys
15
of aprotinin forms salt bridges with conserved negatively charged residues of the omptin active site.
Chlamydia muridarum Induction of Glandular Duct Dilation in Mice
Authors: Sun Xin , Yang Zhangsheng , Zhang Hongbo , Dai Jin , Chen Jianlin , Tang Lingli , Rippentrop Sheena , Xue Min , Zhong Guangming , Wu Ganqiu 
Published: April 2015

ABSTRACT

Although
Chlamydia
-induced hydrosalpinx in women and mice has been used as a surrogate marker for tubal infertility, the medical relevance of nontubal pathologies, such as uterine horn dilation, developed in mice following chlamydial infection remains unclear. We now report that the uterine horn dilation correlates with glandular duct dilation detected microscopically following
Chlamydia muridarum
infection. The dilated glandular ducts pushed the uterine horn lumen to closure or dilation and even broke through the myometrium to develop extrusion outside the uterine horn. The severity scores of uterine horn dilation observed macroscopically correlated well with the number of cross sections of the dilated glandular ducts counted under microscopy. Chlamydial infection was detected in the glandular epithelial cells, potentially leading to inflammation and dilation of the glandular ducts. Direct delivery of
C. muridarum
into the mouse uterus increased both uterine horn/glandular duct dilation and hydrosalpinx. However, the chlamydial plasmid, which is essential for the induction of hydrosalpinx, was not required for the induction of uterine horn/glandular duct dilation. Screening 12 strains of mice for uterine horn dilation following
C. muridarum
infection revealed that B10.D2, C57BL/10J, and C57BL/6J mice were most susceptible, followed by BALB/cJ and A/J mice. Deficiency in host genes involved in immune responses failed to significantly alter the
C. muridarum
induction of uterine horn dilation. Nevertheless, the chlamydial induction of uterine horn/glandular duct dilation may be used to evaluate plasmid-independent pathogenicity of
Chlamydia
in susceptible mice.
Staphylococcus aureus MnhF Mediates Cholate Efflux and Facilitates Survival under Human Colonic Conditions
Authors: Sannasiddappa Thippeswamy H. , Hood Graham A. , Hanson Kevan J. , Costabile Adele , Gibson Glenn R. , Clarke Simon R. 
Published: April 2015

ABSTRACT

Resistance to the innate defenses of the intestine is crucial for the survival and carriage of
Staphylococcus aureus
, a common colonizer of the human gut. Bile salts produced by the liver and secreted into the intestines are one such group of molecules with potent antimicrobial activity. The mechanisms by which
S. aureus
is able to resist such defenses in order to colonize and survive in the human gut are unknown. Here we show that
mnhF
confers resistance to bile salts, which can be abrogated by efflux pump inhibitors. MnhF mediates the efflux of radiolabeled cholic acid both in
S. aureus
and when heterologously expressed in
Escherichia coli
, rendering them resistant. Deletion of
mnhF
attenuated the survival of
S. aureus
in an anaerobic three-stage continuous-culture model of the human colon (gut model), which represents different anatomical areas of the large intestine.
Analysis of the Effects of Cigarette Smoke on Staphylococcal Virulence Phenotypes
Authors: McEachern Elisa K. , Hwang John H. , Sladewski Katherine M. , Nicatia Shari , Dewitz Carola , Mathew Denzil P. , Nizet Victor , Crotty Alexander Laura E. 
Published: April 2015

ABSTRACT

Cigarette smoking is the leading preventable cause of death, disease, and disability worldwide. It is well established that cigarette smoke provokes inflammatory activation and impairs antimicrobial functions of human immune cells. Here we explore whether cigarette smoke likewise affects the virulence properties of an important human pathogen,
Staphylococcus aureus
, and in particular methicillin-resistant
S. aureus
(MRSA), one of the leading causes of invasive bacterial infections. MRSA colonizes the nasopharynx and is thus exposed to inhalants, including cigarette smoke. MRSA exposed to cigarette smoke extract (CSE-MRSA) was more resistant to macrophage killing (4-fold higher survival;
P
< 0.0001). CSE-MRSA demonstrated reduced susceptibility to cell lysis (1.78-fold;
P
= 0.032) and antimicrobial peptide (AMP) (LL-37) killing (MIC, 8 μM versus 4 μM). CSE modified the surface charge of MRSA in a dose-dependent fashion, impairing the binding of particles with charge similar to that of AMPs by 90% (
P
< 0.0001). These changes persisted for 24 h postexposure, suggesting heritable modifications. CSE exposure increased hydrophobicity by 55% (
P
< 0.0001), which complemented findings of increased MRSA adherence and invasion of epithelial cells. CSE induced upregulation of
mprF
, consistent with increased MRSA AMP resistance.
S. aureus
without
mprF
had no change in surface charge upon exposure to CSE.
In vivo
, CSE-MRSA pneumonia induced higher mouse mortality (40% versus 10%) and increased bacterial burden at 8 and 20 h postinfection compared to control MRSA-infected mice (
P
< 0.01). We conclude that cigarette smoke-induced immune resistance phenotypes in MRSA may be an additional factor contributing to susceptibility to infectious disease in cigarette smokers.
Listeria monocytogenes Spreads within the Brain by Actin-Based Intra-Axonal Migration
Authors: Henke Diana , Rupp Sebastian , Gaschen Véronique , Stoffel Michael H. , Frey Joachim , Vandevelde Marc , Oevermann Anna 
Published: April 2015

ABSTRACT

Listeria monocytogenes
rhombencephalitis is a severe progressive disease despite a swift intrathecal immune response. Based on previous observations, we hypothesized that the disease progresses by intra-axonal spread within the central nervous system. To test this hypothesis, neuroanatomical mapping of lesions, immunofluorescence analysis, and electron microscopy were performed on brains of ruminants with naturally occurring rhombencephalitis. In addition, infection assays were performed in bovine brain cell cultures. Mapping of lesions revealed a consistent pattern with a preferential affection of certain nuclear areas and white matter tracts, indicating that
Listeria monocytogenes
spreads intra-axonally within the brain along interneuronal connections. These results were supported by immunofluorescence and ultrastructural data localizing
Listeria monocytogenes
inside axons and dendrites associated with networks of fibrillary structures consistent with actin tails.
In vitro
infection assays confirmed that bacteria were moving within axon-like processes by employing their actin tail machinery. Remarkably,
in vivo
, neutrophils invaded the axonal space and the axon itself, apparently by moving between split myelin lamellae of intact myelin sheaths. This intra-axonal invasion of neutrophils was associated with various stages of axonal degeneration and bacterial phagocytosis. Paradoxically, the ensuing adaxonal microabscesses appeared to provide new bacterial replication sites, thus supporting further bacterial spread. In conclusion, intra-axonal bacterial migration and possibly also the innate immune response play an important role in the intracerebral spread of the agent and hence the progression of listeric rhombencephalitis.
Oxygen-Dependent Cell-to-Cell Variability in the Output of the Escherichia coli Tor Phosphorelay
Authors: Roggiani Manuela , Goulian Mark 
Published: April 2015

ABSTRACT

Escherichia coli
senses and responds to trimethylamine-
N
-oxide (TMAO) in the environment through the TorT-TorS-TorR signal transduction system. The periplasmic protein TorT binds TMAO and stimulates the hybrid kinase TorS to phosphorylate the response regulator TorR through a phosphorelay. Phosphorylated TorR, in turn, activates transcription of the
torCAD
operon, which encodes the proteins required for anaerobic respiration via reduction of TMAO to trimethylamine. Interestingly,
E. coli
respires TMAO in both the presence and absence of oxygen, a behavior that is markedly different from the utilization of other alternative electron acceptors by this bacterium. Here we describe an unusual form of regulation by oxygen for this system. While the average level of
torCAD
transcription is the same for aerobic and anaerobic cultures containing TMAO, the behavior across the population of cells is strikingly different under the two growth conditions. Cellular levels of
torCAD
transcription in aerobic cultures are highly heterogeneous, in contrast to the relatively homogeneous distribution in anaerobic cultures. Thus, oxygen regulates the variance of the output but not the mean for the Tor system. We further show that this oxygen-dependent variability stems from the phosphorelay.

IMPORTANCE
Trimethylamine-
N
-oxide (TMAO) is utilized by numerous bacteria as an electron acceptor for anaerobic respiration. In
E. coli
, expression of the proteins required for TMAO respiration is tightly regulated by a signal transduction system that is activated by TMAO. Curiously, although oxygen is the energetically preferred electron acceptor, TMAO is respired even in the presence of oxygen. Here we describe an interesting and unexpected form of regulation for this system in which oxygen produces highly variable expression of the TMAO utilization proteins across a population of cells without affecting the mean expression of these proteins. To our knowledge, this is the first reported example of a stimulus regulating the variance but not the mean output of a signaling system.
RNase III-Independent Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase via Translational Repression
Authors: Carzaniga Thomas , Dehò Gianni , Briani Federica 
Published: April 2015

ABSTRACT

The complex posttranscriptional regulation mechanism of the
Escherichia coli
pnp
gene, which encodes the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase), involves two endoribonucleases, namely, RNase III and RNase E, and PNPase itself, which thus autoregulates its own expression. The models proposed for
pnp
autoregulation posit that the target of PNPase is a mature
pnp
mRNA previously processed at its 5′ end by RNase III, rather than the primary
pnp
transcript (RNase III-dependent models), and that PNPase activity eventually leads to
pnp
mRNA degradation by RNase E. However, some published data suggest that
pnp
expression may also be regulated through a PNPase-dependent, RNase III-independent mechanism. To address this issue, we constructed isogenic Δ
pnp rnc
+
and Δ
pnp
Δ
rnc
strains with a chromosomal
pnp-lacZ
translational fusion and measured β-galactosidase activity in the absence and presence of PNPase expressed by a plasmid. Our results show that PNPase also regulates its own expression via a reversible RNase III-independent pathway acting upstream from the RNase III-dependent branch. This pathway requires the PNPase RNA binding domains KH and S1 but not its phosphorolytic activity. We suggest that the RNase III-independent autoregulation of PNPase occurs at the level of translational repression, possibly by competition for
pnp
primary transcript between PNPase and the ribosomal protein S1.

IMPORTANCE
In
Escherichia coli
, polynucleotide phosphorylase (PNPase, encoded by
pnp
) posttranscriptionally regulates its own expression. The two models proposed so far posit a two-step mechanism in which RNase III, by cutting the leader region of the
pnp
primary transcript, creates the substrate for PNPase regulatory activity, eventually leading to
pnp
mRNA degradation by RNase E. In this work, we provide evidence supporting an additional pathway for PNPase autogenous regulation in which PNPase acts as a translational repressor independently of RNase III cleavage. Our data make a new contribution to the understanding of the regulatory mechanism of
pnp
mRNA, a process long since considered a paradigmatic example of posttranscriptional regulation at the level of mRNA stability.
The Matrix Reloaded: How Sensing the Extracellular Matrix Synchronizes Bacterial Communities
Authors: Steinberg Nitai , Kolodkin-Gal Ilana 
Published: April 2015

ABSTRACT
In response to chemical communication, bacterial cells often organize themselves into complex multicellular communities that carry out specialized tasks. These communities are frequently referred to as biofilms, which involve the collective behavior of different cell types. Like cells of multicellular eukaryotes, the biofilm cells are surrounded by self-produced polymers that constitute the extracellular matrix (ECM), which binds them to each other and to the surface. In multicellular eukaryotes, it has been evident for decades that cell-ECM interactions control multiple cellular processes during development. While cells both in biofilms and in multicellular eukaryotes are surrounded by ECM and activate various genetic programs, until recently it has been unclear whether cell-ECM interactions are recruited in bacterial communicative behaviors. In this review, we describe the examples reported thus far for ECM involvement in control of cell behavior throughout the different stages of biofilm formation. The studies presented in this review have provided a newly emerging perspective of the bacterial ECM as an active player in regulation of biofilm development.
Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis
Authors: Black Katherine A. , Dos Santos Patricia C. 
Published: April 2015

ABSTRACTThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U inEscherichia colirequires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coliMnmA adenylates and subsequently thiolates tRNA to form the s2U modification.Bacillus subtilislacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO, directly adjacent tomnmA. The genomic synteny ofyrvOandmnmAcombined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that theB. subtilisYrvO and MnmA are sufficient for s2U biosynthesis. A conditionalB. subtilisknockout strain showed that s2U abundance correlates with MnmA expression, andin vivocomplementation studies inE. coliIscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis.In vitroexperiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilisproteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coliΔiscSand ΔmnmAstrains associated with s2U depletion is recovered byB. subtilis yrvOandmnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U inEscherichia colirequires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilisand most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmAcoding sequence is located adjacent toyrvO, encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilisYrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2U biosynthesis in a pathway independent of the one used inE. coli.
Targeted Knockout of the Rickettsia rickettsii OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System
Authors: Noriea Nicholas F. , Clark Tina R. , Hackstadt Ted 
Published: April 2015

ABSTRACT

Strains of
Rickettsia rickettsii
, the causative agent of Rocky Mountain spotted fever (RMSF), differ dramatically in virulence despite >99% genetic homology. Spotted fever group (SFG) rickettsiae produce two immunodominant outer membrane proteins, rickettsial OmpA (rOmpA) and rOmpB, which are conserved throughout the SFG and thought to be fundamental to pathogenesis. rOmpA is present in all virulent strains of
R. rickettsii
but is not produced in the only documented avirulent strain, Iowa, due to a premature stop codon. Here we report the creation of an isogenic
ompA
mutant in the highly virulent strain Sheila Smith by insertion of intronic RNA to create a premature stop codon 312 bp downstream of the 6,747-bp open reading frame initiation site (int312). Targeted insertion was accomplished using an LtrA group II intron retrohoming system. Growth and entry rates of Sheila Smith
ompA
::int312 in Vero cells remained comparable to those of the wild type. Virulence was assessed in a guinea pig model by challenge with 100 PFU of either
ompA
::int312 Sheila Smith or the wild type, but no significant difference in either fever peak (40.5°C) or duration (8 days) were shown between the wild type and the knockout. The ability to disrupt genes in a site-specific manner using an LtrA group II intron system provides an important new tool for evaluation of potential virulence determinants in rickettsial disease research.

IMPORTANCE
R. rickettsii
rOmpA is an immunodominant outer membrane autotransporter conserved in the spotted fever group. Previous studies and genomic comparisons suggest that rOmpA is involved in adhesion and may be critical for virulence. Little information is available for rickettsial virulence factors in an isogenic background, as limited systems for targeted gene disruption are currently available. Here we describe the creation of an rOmpA knockout by insertion of a premature stop codon into the 5′ end of the open reading frame using a group II intron system. An isogenic rOmpA knockout mutation in the highly virulent Sheila Smith strain did not cause attenuation in a guinea pig model of infection, and no altered phenotype was observed in cell culture. We conclude that rOmpA is not critical for virulence in a guinea pig model but may play a role in survival or transmission from the tick vector.