bluuebunny/crossref_metadata_2025_split huggingface dataset:
Trafficking through the Late Endosome Significantly Impacts Candida albicans Tolerance of the Azole Antifungals
Authors: Luna-Tapia Arturo , Kerns Morgan E. , Eberle Karen E. , Jursic Branko S. , Palmer Glen E. 
Published: February 2015

ABSTRACT

The azole antifungals block ergosterol biosynthesis by inhibiting lanosterol demethylase (Erg11p). The resulting depletion of cellular ergosterol and the accumulation of “toxic” sterol intermediates are both thought to compromise plasma membrane function. However, the effects of ergosterol depletion upon the function of intracellular membranes and organelles are not well described. The purpose of this study was to characterize the effects of azole treatment upon the integrity of the
Candida albicans
vacuole and to determine whether, in turn, vacuolar trafficking influences azole susceptibility. Profound fragmentation of the
C. albicans
vacuole can be observed as an early consequence of azole treatment, and it precedes significant growth inhibition. In addition, a
C. albicans
vps21
Δ/Δ mutant, blocked in membrane trafficking through the late endosomal prevacuolar compartment (PVC), is able to grow significantly more than the wild type in the presence of several azole antifungals under standard susceptibility testing conditions. Furthermore, the
vps21
Δ/Δ mutant is able to grow despite the depletion of cellular ergosterol. This phenotype resembles an exaggerated form of “trailing growth” that has been described for some clinical isolates. In contrast, the
vps21
Δ/Δ mutant is hypersensitive to drugs that block alternate steps in ergosterol biosynthesis. On the basis of these results, we propose that endosomal trafficking defects may lead to the cellular “redistribution” of the sterol intermediates that accumulate following inhibition of ergosterol biosynthesis. Furthermore, the destination of these intermediates, or the precise cellular compartments in which they accumulate, may be an important determinant of their toxicity and thus ultimately antifungal efficacy.
Variants of β-Lactamase KPC-2 That Are Resistant to Inhibition by Avibactam
Authors: Papp-Wallace Krisztina M. , Winkler Marisa L. , Taracila Magdalena A. , Bonomo Robert A. 
Published: February 2015

ABSTRACT

KPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling.
Escherichia coli
DH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; the
k
2
/
K
ratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M
−1
s
−1
to 1.2 M
−1
s
−1
). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R220 contribute significantly to the mechanism of avibactam inactivation of KPC-2. Fortunately, the emergence of S130G, K234R, and R220M variants of KPC in the clinic should not result in failure of ceftazidime-avibactam, as the ceftazidime partner is potent against
E. coli
DH10B strains possessing all of these variants.
Moxifloxacin Pharmacokinetic Profile and Efficacy Evaluation in Empiric Treatment of Community-Acquired Pneumonia
Authors: Öbrink-Hansen Kristina , Hardlei Tore Forsingdal , Brock Birgitte , Jensen-Fangel Søren , Kragh Thomsen Marianne , Petersen Eskild , Kreilgaard Mads 
Published: February 2015

ABSTRACT

When antimicrobials are used empirically, pathogen MICs equal to clinical breakpoints or epidemiological cutoff values must be considered. This is to ensure that the most resistant pathogen subpopulation is appropriately targeted to prevent emergence of resistance. Accordingly, we determined the pharmacokinetic (PK) profile of moxifloxacin at 400 mg/day in 18 patients treated empirically for community-acquired pneumonia. We developed a population pharmacokinetic model to assess the potential efficacy of moxifloxacin and to simulate the maximal MICs for which recommended pharmacokinetic-pharmacodynamic (PK-PD) estimates are obtained. Moxifloxacin plasma concentrations were determined the day after therapy initiation using ultra-high-performance liquid chromatography. Peak drug concentrations (
C
max
) and area under the free drug concentration-time curve from 0 to 24 h (
f
AUC
0–24
) values predicted for each patient were evaluated against epidemiological cutoff MIC values for
Streptococcus pneumoniae
,
Haemophilus influenzae
, and
Legionella pneumophila
. PK-PD targets adopted were a
C
max
/MIC of ≥12.2 for all pathogens, an
f
AUC
0–24
/MIC of >34 for
S. pneumoniae
, and an
f
AUC
0–24
/MIC of >75 for
H. influenzae
and
L. pneumophila
. Individual predicted estimates for
C
max
/MIC and
f
AUC
0–24
/MIC as well as simulated maximal MICs resulting in target attainment for oral and intravenous administration of the drug were suitable for
S. pneumoniae
and
H. influenzae
but not for
L. pneumophila
. These results indicate that caution must be taken when moxifloxacin is used as monotherapy to treat community-acquired pneumonia caused by
L. pneumophila
. In conclusion, this report reveals key information relevant to the empirical treatment of community-acquired pneumonia while highlighting the robust and flexible nature of this population pharmacokinetic model to predict therapeutic success. (Clinical Trials Registration no. NCT01983839.)
Cyclophilin and NS5A Inhibitors, but Not Other Anti-Hepatitis C Virus (HCV) Agents, Preclude HCV-Mediated Formation of Double-Membrane-Vesicle Viral Factories
Authors: Chatterji Udayan , Bobardt Michael , Tai Andrew , Wood Malcolm , Gallay Philippe A. 
Published: February 2015

ABSTRACT
Although the mechanisms of action (MoA) of nonstructural protein 3 inhibitors (NS3i) and NS5B inhibitors (NS5Bi) are well understood, the MoA of cyclophilin inhibitors (CypI) and NS5A inhibitors (NS5Ai) are not fully defined. In this study, we examined whether CypI and NS5Ai interfere with hepatitis C virus (HCV) RNA synthesis of replication complexes (RCs) or with an earlier step of HCV RNA replication, the creation of double-membrane vesicles (DMVs) essential for HCV RNA replication. In contrast to NS5Bi, both CypI and NS5Ai do not block HCV RNA synthesis by way of RCs, suggesting that they exert their antiviral activity prior to the establishment of enzymatically active RCs. We found that viral replication is not a precondition for DMV formation, since the NS3-NS5B polyprotein or NS5A suffices to create DMVs. Importantly, only CypI and NS5Ai, but not NS5Bi, mir-122, or phosphatidylinositol-4 kinase IIIα (PI4KIIIα) inhibitors, prevent NS3-NS5B-mediated DMV formation. NS3-NS5B was unable to create DMVs in cyclophilin A (CypA) knockdown (KD) cells. We also found that the isomerase activity of CypA is absolutely required for DMV formation. This not only suggests that NS5A and CypA act in concert to build membranous viral factories but that CypI and NS5Ai mediate their early anti-HCV effects by preventing the formation of organelles, where HCV replication is normally initiated. This is the first investigation to examine the effect of a large panel of anti-HCV agents on DMV formation, and the results reveal that CypI and NS5Ai act at the same membranous web biogenesis step of HCV RNA replication, thus indicating a new therapeutic target of chronic hepatitis C.
Combination of the R263K and M184I/V Resistance Substitutions against Dolutegravir and Lamivudine Decreases HIV Replicative Capacity
Authors: Singhroy Diane N. , Wainberg Mark A. , Mesplède Thibault 
Published: February 2015

ABSTRACT
We investigated the effect of combining the dolutegravir-specific R263K integrase resistance substitution with either M184I or M184V, two reverse transcriptase drug resistance substitutions that are frequently detected in individuals failing therapeutic regimens containing either lamivudine or emtricitabine. The presence of R263K and M184I/V in a single virus resulted in substantial further decreases in the viral replicative capacity compared to that in the presence of single substitutions alone.
Dermatophytes Activate Skin Keratinocytes via Mitogen-Activated Protein Kinase Signaling and Induce Immune Responses
Authors: Achterman Rebecca R. , Moyes David L. , Thavaraj Selvam , Smith Adam R. , Blair Kris M. , White Theodore C. , Naglik Julian R. 
Published: February 2015

ABSTRACT

Dermatophytes cause superficial and cutaneous fungal infections in immunocompetent hosts and invasive disease in immunocompromised hosts. However, the host mechanisms that regulate innate immune responses against these fungi are largely unknown. Here, we utilized commercially available epidermal tissues and primary keratinocytes to assess (i) damage induction by anthropophilic, geophilic, and zoophilic dermatophyte strains and (ii) the keratinocyte signaling pathways, transcription factors, and proinflammatory responses induced by a representative dermatophyte,
Trichophyton equinum
. Initially, five dermatophyte species were tested for their ability to invade, cause tissue damage, and induce cytokines, with
Microsporum gypseum
inducing the greatest level of damage and cytokine release. Using
T. equinum
as a representative dermatophyte, we found that the mitogen-activated protein kinase (MAPK) pathways were predominantly affected, with increased levels of phospho-p38 and phospho-Jun N-terminal protein kinase (JNK) but decreased levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2). Notably, the NF-κB and PI3K pathways were largely unaffected.
T. equinum
also significantly increased expression of the AP-1-associated transcription factor, c-Fos, and the MAPK regulatory phosphatase, MKP1. Importantly, the ability of
T. equinum
to invade, cause tissue damage, activate signaling and transcription factors, and induce proinflammatory responses correlated with germination, indicating that germination may be important for dermatophyte virulence and host immune activation.
Role of Copper Efflux in Pneumococcal Pathogenesis and Resistance to Macrophage-Mediated Immune Clearance
Authors: Johnson Michael D. L. , Kehl-Fie Thomas E. , Klein Roger , Kelly Jacqueline , Burnham Corinna , Mann Beth , Rosch Jason W. 
Published: February 2015

ABSTRACT

In bacteria, the intracellular levels of metals are mediated by tightly controlled acquisition and efflux systems. This is particularly true of copper, a trace element that is universally toxic in excess. During infection, the toxic properties of copper are exploited by the mammalian host to facilitate bacterial clearance. To better understand the role of copper during infection, we characterized the contribution of the
cop
operon to copper homeostasis and virulence in
Streptococcus pneumoniae
. Deletion of either the exporter, encoded by
copA
, or the chaperone, encoded by
cupA
, led to hypersensitivity to copper stress. We further demonstrated that loss of the copper exporter encoded by
copA
led to decreased virulence in pulmonary, intraperitoneal, and intravenous models of infection. Deletion of
copA
resulted in enhanced macrophage-mediated bacterial clearance
in vitro
. The attenuation phenotype of the
copA
mutant in the lung was found to be dependent on pulmonary macrophages, underscoring the importance of copper efflux in evading immune defenses. Overall, these data provide insight into the role of the
cop
operon in pneumococcal pathogenesis.
Two-Tiered Histidine Kinase Pathway Involved in Heat Shock and Salt Sensing in the General Stress Response of Sphingomonas melonis Fr1
Authors: Kaczmarczyk Andreas , Hochstrasser Ramon , Vorholt Julia A. , Francez-Charlot Anne 
Published: February 2015

ABSTRACT

The general stress response (GSR) allows bacteria to monitor and defend against a broad set of unrelated, adverse environmental conditions. In
Alphaproteobacteria
, the key step in GSR activation is phosphorylation of the response regulator PhyR. In
Sphingomonas melonis
Fr1, seven PhyR-activating kinases (Paks), PakA to PakG, are thought to directly phosphorylate PhyR under different stress conditions, but the nature of the activating signals remains obscure. PakF, a major sensor of NaCl and heat shock, lacks a putative sensor domain but instead harbors a single receiver (REC) domain (PakF
REC
) N-terminal to its kinase catalytic core. Such kinases are called “hybrid response regulators” (HRRs). How HRRs are able to perceive signals in the absence of a true sensor domain has remained largely unexplored. In the present work, we show that stresses are actually sensed by another kinase, KipF (kinase of PakF), which phosphorylates PakF
REC
and thereby activates PakF. KipF is a predicted transmembrane kinase, harboring a periplasmic CHASE3 domain flanked by two transmembrane helices in addition to its cytoplasmic kinase catalytic core. We demonstrate that KipF senses different salts through its CHASE3 domain but is not a sensor of general osmotic stress. While salt sensing depends on the CHASE3 domain, heat shock sensing does not, suggesting that these stresses are perceived by different mechanisms. In summary, our results establish a two-tiered histidine kinase pathway involved in activation of the GSR in
S. melonis
Fr1 and provide the first experimental evidence for the so far uncharacterized CHASE3 domain as a salt sensor.

IMPORTANCE
Hybrid response regulators (HRRs) represent a particular class of histidine kinases harboring an N-terminal receiver (REC) domain instead of a true sensor domain. This suggests that the actual input for HRRs may be phosphorylation of the REC domain. In the present study, we addressed this question by using the HRR PakF. Our results suggest that PakF is activated through phosphorylation of its REC domain and that this is achieved by another kinase, KipF. KipF senses heat shock and salt stress, with the latter requiring the periplasmic CHASE3 domain. This work not only suggests that HRRs work in two-tiered histidine kinase pathways but also provides the first experimental evidence for a role of the so far uncharacterized CHASE3 domain in salt sensing.
The α-Glucan Phosphorylase MalP of Corynebacterium glutamicum Is Subject to Transcriptional Regulation and Competitive Inhibition by ADP-Glucose
Authors: Clermont Lina , Macha Arthur , Müller Laura M. , Derya Sami M. , von Zaluskowski Philipp , Eck Alexander , Eikmanns Bernhard J. , Seibold Gerd M. 
Published: February 2015

ABSTRACT

α-Glucan phosphorylases contribute to degradation of glycogen and maltodextrins formed in the course of maltose metabolism in bacteria. Accordingly, bacterial α-glucan phosphorylases are classified as either glycogen or maltodextrin phosphorylase, GlgP or MalP, respectively. GlgP and MalP enzymes follow the same catalytic mechanism, and thus their substrate spectra overlap; however, they differ in their regulation: GlgP genes are constitutively expressed and the enzymes are controlled on the activity level, whereas expression of MalP genes are transcriptionally controlled in response to the carbon source used for cultivation. We characterize here the modes of control of the α-glucan phosphorylase MalP of the Gram-positive
Corynebacterium glutamicum
. In accordance to the proposed function of the
malP
gene product as MalP, we found transcription of
malP
to be regulated in response to the carbon source. Moreover,
malP
transcription is shown to depend on the growth phase and to occur independently of the cell glycogen content. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. Since the latter is considered a typical feature of GlgPs, we propose that
C. glutamicum
MalP acts as both maltodextrin and glycogen phosphorylase and, based on these findings, we question the current system for classification of bacterial α-glucan phosphorylases.

IMPORTANCE
Bacterial α-glucan phosphorylases have been classified conferring to their purpose as either glycogen or maltodextrin phosphorylases. We found transcription of
malP
in
C. glutamicum
to be regulated in response to the carbon source, which is recognized as typical for maltodextrin phosphorylases. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. The latter is considered a typical feature of GlgPs. These findings, taken together, suggest that
C. glutamicum
MalP is the first α-glucan phosphorylase that does not fit into the current system for classification of bacterial α-glucan phosphorylases and exemplifies the complex mechanisms underlying the control of glycogen content and maltose metabolism in this model organism.
The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene,
adhE
, Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum
Authors: Lo Jonathan , Zheng Tianyong , Hon Shuen , Olson Daniel G. , Lynd Lee R. 
Published: February 2015

ABSTRACT

Thermoanaerobacterium saccharolyticum
and
Clostridium thermocellum
are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene,
adhE
, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the
adhE
gene in
C. thermocellum
and
T. saccharolyticum
. Deletion of
adhE
reduced ethanol production by >95% in both
T. saccharolyticum
and
C. thermocellum
, confirming that
adhE
is necessary for ethanol formation in both organisms. In both
adhE
deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In
T. saccharolyticum
, the
adhE
deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the
T. saccharolyticum
parent strain but did not increase activity in the
adhE
deletion strain, suggesting that ALDH activity was inhibited. In
C. thermocellum
, the
adhE
deletion strain lost >90% of ALDH and ADH activity in cell extracts. The
C. thermocellum
adhE
deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase.

IMPORTANCE
Thermoanaerobacterium saccharolyticum
and
Clostridium thermocellum
are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be responsible for alcohol formation. This study explores the inactivation of
adhE
, a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion of
adhE
reduced ethanol production by >95% in both
T. saccharolyticum
and
C. thermocellum
, confirming that
adhE
is necessary for ethanol formation in both organisms. In strains without
adhE
, we note changes in biochemical activity, product formation, and growth.
Violence, self-harm and drug or alcohol misuse in adolescents admitted to hospitals in England for injury: a retrospective cohort study
Authors: Herbert Annie , Gilbert Ruth , González-Izquierdo Arturo , Li Leah 
Published: February 2015

ObjectivesOf adolescents in the general population in England, we aimed to determine (1) the proportion that has an emergency admission to hospital for injury related to adversity (violence, self-harm or drug or alcohol misuse) and (2) the risk of recurrent emergency admissions for injury in adolescents admitted with adversity-related injury compared with those admitted with accident-related injury only.DesignWe used longitudinally linked administrative hospital data (Hospital Episode Statistics) to identify participants aged 10–19 years with emergency admissions for injury (including day cases lasting more than 4 h) in England in 1998–2011. We used the Office for National Statistics mid-year estimates for population denominators.ResultsApproximately 4.3% (n=141 248) of adolescents in the general population (n=3 254 046) had one or more emergency admissions for adversity-related injury (girls 4.6%, boys 4.1%), accounting for 50% of all emergency admissions for injury in girls and 29.1% in boys. Admissions for self-harm or drug or alcohol misuse commonly occurred in the same girls and boys. Recurrent emergency admissions for injury were more common in adolescents with adversity-related injury (girls 17.3%, boys 16.5%) than in those with accident-related injury only (girls 4.7%, boys 7.4%), particularly for adolescents with adversity-related injury related to multiple types of adversity (girls 21.1%, boys 24.2%).ConclusionsHospital-based interventions should be developed to reduce the risk of future injury in adolescents admitted for adversity-related injury.