bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bioluminescent Imaging Reveals Novel Patterns of Colonization and Invasion in Systemic Escherichia coli K1 Experimental Infection in the Neonatal Rat
Authors: Witcomb Luci A. , Collins James W. , McCarthy Alex J. , Frankel Gadi , Taylor Peter W. 
Published: September 2015

ABSTRACT

Key features of
Escherichia coli
K1-mediated neonatal sepsis and meningitis, such as a strong age dependency and development along the gut-mesentery-blood-brain course of infection, can be replicated in the newborn rat. We examined temporal and spatial aspects of
E. coli
K1 infection following initiation of gastrointestinal colonization in 2-day-old (P2) rats after oral administration of
E. coli
K1 strain A192PP and a virulent bioluminescent derivative,
E. coli
A192PP-
lux
2. A combination of bacterial enumeration in the major organs, two-dimensional bioluminescence imaging, and three-dimensional diffuse light imaging tomography with integrated micro-computed tomography indicated multiple sites of colonization within the alimentary canal; these included the tongue, esophagus, and stomach in addition to the small intestine and colon. After invasion of the blood compartment, the bacteria entered the central nervous system, with restricted colonization of the brain, and also invaded the major organs, in line with increases in the severity of symptoms of infection. Both keratinized and nonkeratinized surfaces of esophagi were colonized to a considerably greater extent in susceptible P2 neonates than in corresponding tissues from infection-resistant 9-day-old rat pups; the bacteria appeared to damage and penetrate the nonkeratinized esophageal epithelium of infection-susceptible P2 animals, suggesting the esophagus represents a portal of entry for
E. coli
K1 into the systemic circulation. Thus, multimodality imaging of experimental systemic infections in real time indicates complex dynamic patterns of colonization and dissemination that provide new insights into the
E. coli
K1 infection of the neonatal rat.
The Candida albicans ATO Gene Family Promotes Neutralization of the Macrophage Phagolysosome
Authors: Danhof Heather A. , Lorenz Michael C. 
Published: September 2015

ABSTRACTCandida albicansis an opportunistic human fungal pathogen that causes a variety of diseases, ranging from superficial mucosal to life-threatening systemic infections, the latter particularly in patients with defects in innate immune function.C. albicanscells phagocytosed by macrophages undergo a dramatic change in their metabolism in which amino acids are a key nutrient. We have shown that amino acid catabolism allows the cell to neutralize the phagolysosome and initiate hyphal growth. We show here that members of the 10-geneATOfamily, which are induced by phagocytosis or the presence of amino acids in an Stp2-dependent manner and encode putative acetate or ammonia transporters, are important effectors of this pH changein vitroand in macrophages. When grown with amino acids as the sole carbon source, the deletion ofATO5or the expression of a dominant-negativeATO1G53Dallele results in a delay in alkalinization, a defect in hyphal formation, and a reduction in the amount of ammonia released from the cell. These strains also form fewer hyphae after phagocytosis, have a reduced ability to escape macrophages, and reside in more acidic phagolysosomal compartments than wild-type cells. Furthermore, overexpression of many of the 10ATOgenes accelerates ammonia release, and anato5Δ ATO1G53Ddouble mutant strain has additive alkalinization and ammonia release defects. Taken together, these results indicate that the Ato protein family is a key mediator of the metabolic changes that allowC. albicansto overcome the macrophage innate immunity barrier.
Loss of NADH Oxidase Activity in Streptococcus mutans Leads to Rex-Mediated Overcompensation in NAD
+
Regeneration by Lactate Dehydrogenase
Authors: Baker J. L. , Derr A. M. , Faustoferri R. C. , Quivey R. G. 
Published: September 2015

ABSTRACT

Previous studies of the oral pathogen
Streptococcus mutans
have determined that this Gram-positive facultative anaerobe mounts robust responses to both acid and oxidative stresses. The water-forming NADH oxidase (Nox; encoded by
nox
) is thought to be critical for the regeneration of NAD
+
, for use in glycolysis, and for the reduction of oxygen, thereby preventing the formation of damaging reactive oxygen species. In this study, the free NAD
+
/NADH ratio in a
nox
deletion strain (Δ
nox
) was discovered to be remarkably higher than that in the parent strain, UA159, when the strains were grown in continuous culture. This unanticipated result was explained by significantly elevated lactate dehydrogenase (Ldh; encoded by
ldh
) activity and
ldh
transcription in the Δ
nox
strain, which was mediated in part by the redox-sensing regulator Rex. cDNA microarray analysis of
S. mutans
cultures exposed to simultaneous acid stress (growth at a low pH) and oxidative stress (generated through the deletion of
nox
or the addition of exogenous oxygen) revealed a stress response synergistically heightened over that with either stress alone. In the Δ
nox
strain, this elevated stress response included increased glucose phosphoenolpyruvate phosphotransferase system (PTS) activity, which appeared to be due to elevated
manL
transcription, mediated in part, like elevated
ldh
transcription, by Rex. While the Δ
nox
strain does possess a membrane composition different from that of the parent strain, it did not appear to have defects in either membrane permeability or ATPase activity. However, the altered transcriptome and metabolome of the Δ
nox
strain were sufficient to impair its ability to compete with commensal peroxigenic oral streptococci during growth under aerobic conditions.

IMPORTANCE
Streptococcus mutans
is an oral pathogen whose ability to outcompete commensal oral streptococci is strongly linked to the formation of dental caries. Previous work has demonstrated that the
S. mutans
water-forming NADH oxidase is critical for both carbon metabolism and the prevention of oxidative stress. The results of this study show that upregulation of lactate dehydrogenase, mediated through the redox sensor Rex, overcompensates for the loss of
nox
. Additionally,
nox
deletion led to the upregulation of mannose and glucose transport, also mediated through Rex. Importantly, the loss of
nox
rendered
S. mutans
defective in its ability to compete directly with two species of commensal streptococci, suggesting a role for
nox
in the pathogenic potential of this organism.
HK022 Nun Requires Arginine-Rich Motif Residues Distinct from λ N
Authors: Tawk Caroline S. , Ghattas Ingrid R. , Smith Colin A. 
Published: September 2015

ABSTRACT
Bacteriophage λ N protein binds boxB RNA hairpins in the nut (N utilization) sites of immediate early λ transcripts and interacts with host factors to suppress transcriptional termination at downstream terminators. In opposition to λ N, the Nun protein of HK022 binds the boxBs of coinfecting λ transcripts, interacts with a similar or identical set of host factors, and terminates transcription to suppress λ replication. Comparison of N-boxB and Nun-boxB nuclear magnetic resonance (NMR) structural models suggests similar interactions, though limited mutagenesis of Nun is available. Here, libraries of Nun's arginine-rich motif (ARM) were screened for the ability to exclude λ coinfection, and mutants were assayed for Nun termination with a boxB plasmid reporter system. Several Nun ARM residues appear to be immutable: Asp26, Arg28, Arg29, Arg32, Trp33, and Arg36. Asp26 and Trp33 appear to be unable to contact boxB and are not found at equivalent positions in λ N ARM. To understand if the requirement of Asp26, Trp33, and Arg36 indicated differences between HK022 Nun termination and λ N antitermination complexes, the same Nun libraries were fused to the activation domain of λ N and screened for clones able to complement N-deficient λ. Mutants were assayed for N antitermination. Surprisingly, Asp26 and Trp33 were still essential when Nun ARM was fused to N. Docking suggests that Nun ARM contacts a hydrophobic surface of the NusG carboxy-terminal domain containing residues necessary for Nun function. These findings indicate that Nun ARM relies on distinct contacts in its ternary complex and illustrate how protein-RNA recognition can evolve new regulatory functions.

IMPORTANCE
λ N protein interacts with host factors to allow λ nut-containing transcripts to elongate past termination signals. A competing bacteriophage, HK022, expresses Nun protein, which causes termination of λ nut transcripts. λ N and HK022 Nun use similar arginine-rich motifs (ARMs) to bind the same boxB RNAs in nut transcripts. Screening libraries of Nun ARM mutants, both in HK022 Nun and in a λ N fusion, revealed amino acids essential to Nun that could bind one or more host factors. Docking suggests that NusG, which is present in both Nun termination and N antitermination, is a plausible partner. These findings could help understand how transcription elongation is regulated and illustrate how subtle differences allow ARMs to evolve new regulatory functions.
Genetic, Genomic, and Transcriptomic Studies of Pyruvate Metabolism in Methanosarcina barkeri Fusaro
Authors: López Muñoz Madeline M. , Schönheit Peter , Metcalf William W. 
Published: September 2015

ABSTRACT

Pyruvate, a central intermediate in the carbon fixation pathway of methanogenic archaea, is rarely used as an energy source by these organisms. The sole exception to this rule is a genetically uncharacterized
Methanosarcina barkeri
mutant capable of using pyruvate as a sole energy and carbon source (the Pyr
+
phenotype). Here, we provide evidence that suggests that the Pyr
+
mutant is able to metabolize pyruvate by overexpressing pyruvate ferredoxin oxidoreductase (
por
) and mutating genes involved in central carbon metabolism. Genomic analysis showed that the Pyr
+
strain has two mutations localized to Mbar_A1588, the biotin protein ligase subunit of the pyruvate carboxylase (
pyc
) operon, and Mbar_A2165, a putative transcriptional regulator. Mutants expressing the Mbar_A1588 mutation showed no growth defect compared to the wild type (WT), yet the strains lacked
pyc
activity. Recreation of the Mbar_A2165 mutation resulted in a 2-fold increase of Por activity and gene expression, suggesting a role in
por
transcriptional regulation. Further transcriptomic analysis revealed that Pyr
+
strains also overexpress the gene encoding phosphoenolpyruvate carboxylase, indicating the presence of a previously uncharacterized route for synthesizing oxaloacetate in
M. barkeri
and explaining the unimpaired growth in the absence of Pyc. Surprisingly, stringent repression of the
por
operon was lethal, even when the media were supplemented with pyruvate and/or Casamino Acids, suggesting that
por
plays an unidentified essential function in
M. barkeri
.

IMPORTANCE
The work presented here reveals a complex interaction between anabolic and catabolic pathways involving pyruvate metabolism in
Methanosarcina barkeri
Fusaro. Among the unexpected findings were an essential role for the enzyme pyruvate-ferredoxin oxidoreductase and an alternate pathway for synthesis of oxaloacetate. These results clarify the mechanism of methanogenic catabolism of pyruvate and expand our understanding of carbon assimilation in methanogens.
The KdpD Sensor Kinase of Escherichia coli Responds to Several Distinct Signals To Turn on Expression of the Kdp Transport System
Authors: Epstein Wolfgang 
Published: September 2015

ABSTRACT

Kdp, one of three saturable K
+
uptake systems in
Escherichia coli
, is the system with the highest affinity for K
+
and the only one whose expression is strongly controlled by medium K
+
concentration. Expression is controlled by a two-component system of KdpD, the sensor kinase, and KdpE, the response regulator. There is general agreement that expression occurs when the growth rate of cells begins to become limited by K
+
availability. How K
+
limitation results in expression has been controversial. Studying the roles of the major components of the growth medium shows that KdpD senses at least two distinct signals inside the cell, those of Na
+
and NH
4
+
, and it probably senses other monovalent cations in the cell. KdpD does not sense turgor.

IMPORTANCE
The expression of the Kdp K
+
transport system of
E. coli
occurs when cells become limited in their growth rate by the availability of K
+
. Cells sense limited K
+
and try to compensate by taking up other monovalent cations, particularly Na
+
and NH
4
+
. These cations are sensed in the cytoplasm by the KdpD response regulator, presumably to stimulate its kinase activity. It is shown that KdpD does not sense turgor, as was suggested earlier.
New Elements To Consider When Modeling the Hazards Associated with Botulinum Neurotoxin in Food
Authors: Ihekwaba Adaoha E. C. , Mura Ivan , Malakar Pradeep K. , Walshaw John , Peck Michael W. , Barker G. C. 
Published: September 2015

ABSTRACTBotulinum neurotoxins (BoNTs) produced by the anaerobic bacteriumClostridium botulinumare the most potent biological substances known to mankind. BoNTs are the agents responsible for botulism, a rare condition affecting the neuromuscular junction and causing a spectrum of diseases ranging from mild cranial nerve palsies to acute respiratory failure and death. BoNTs are a potential biowarfare threat and a public health hazard, since outbreaks of foodborne botulism are caused by the ingestion of preformed BoNTs in food. Currently, mathematical models relating to the hazards associated withC. botulinum, which are largely empirical, make major contributions to botulinum risk assessment. Evaluated using statistical techniques, these models simulate the response of the bacterium to environmental conditions. Though empirical models have been successfully incorporated into risk assessments to support food safety decision making, this process includes significant uncertainties so that relevant decision making is frequently conservative and inflexible. Progression involves encoding into the models cellular processes at a molecular level, especially the details of the genetic and molecular machinery. This addition drives the connection between biological mechanisms and botulism risk assessment and hazard management strategies. This review brings together elements currently described in the literature that will be useful in building quantitative models ofC. botulinumneurotoxin production. Subsequently, it outlines how the established form of modeling could be extended to include these new elements. Ultimately, this can offer further contributions to risk assessments to support food safety decision making.
RbsR Activates Capsule but Represses the
rbsUDK
Operon in Staphylococcus aureus
Authors: Lei Mei G. , Lee Chia Y. 
Published: September 2015

ABSTRACT

Staphylococcus aureus
capsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of the
cap
operon. A 10-bp inverted repeat (IR) located 13 bp upstream of the −35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of the
cap
promoter. To search for potential proteins which directly interact with the
cap
promoter region (P
cap
), we directly analyzed the proteins interacting with the P
cap
DNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulate
cap
gene expression by specifically binding to the
cap
promoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed that
rbsR
was directly controlled by SigB and that RbsR was a repressor of the
rbsUDK
operon, involved in ribose uptake and phosphorylation. The repression of
rbsUDK
by RbsR could be derepressed by
d
-ribose. However,
d
-ribose did not affect RbsR activation of capsule.

IMPORTANCE
Staphylococcus aureus
is an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression of
rbsR
. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits the
rbs
operon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence in
S. aureus
. Thus, this study further advances our understanding of staphylococcal virulence regulation.
Growth-Phase-Specific Modulation of Cell Morphology and Gene Expression by an Archaeal Histone Protein
Authors: Dulmage Keely A. , Todor Horia , Schmid Amy K. 
Published: September 2015

ABSTRACT

In all three domains of life, organisms use nonspecific DNA-binding proteins to compact and organize the genome as well as to regulate transcription on a global scale. Histone is the primary eukaryotic nucleoprotein, and its evolutionary roots can be traced to the archaea. However, not all archaea use this protein as the primary DNA-packaging component, raising questions regarding the role of histones in archaeal chromatin function. Here, quantitative phenotyping, transcriptomic, and proteomic assays were performed on deletion and overexpression mutants of the sole histone protein of the hypersaline-adapted haloarchaeal model organism
Halobacterium salinarum
. This protein is highly conserved among all sequenced haloarchaeal species and maintains hallmark residues required for eukaryotic histone functions. Surprisingly, despite this conservation at the sequence level, unlike in other archaea or eukaryotes,
H. salinarum
histone is required to regulate cell shape but is not necessary for survival. Genome-wide expression changes in histone deletion strains were global, significant but subtle in terms of fold change, bidirectional, and growth phase dependent. Mass spectrometric proteomic identification of proteins from chromatin enrichments yielded levels of histone and putative nucleoid-associated proteins similar to those of transcription factors, consistent with an open and transcriptionally active genome. Taken together, these data suggest that histone in
H. salinarum
plays a minor role in DNA compaction but important roles in growth-phase-dependent gene expression and regulation of cell shape. Histone function in haloarchaea more closely resembles a regulator of gene expression than a chromatin-organizing protein like canonical eukaryotic histone.

IMPORTANCE
Histones comprise the major protein component of eukaryotic chromatin and are required for both genome packaging and global regulation of expression. The current paradigm maintains that archaea whose genes encode histone also use these proteins to package DNA. In contrast, here we demonstrate that the sole histone encoded in the genome of the salt-adapted archaeon
Halobacterium salinarum
is both unessential and unlikely to be involved in DNA compaction despite conservation of residues important for eukaryotic histones. Rather,
H. salinarum
histone is required for global regulation of gene expression and cell shape. These data are consistent with the hypothesis that
H. salinarum
histone, strongly conserved across all other known salt-adapted archaea, serves a novel role in gene regulation and cell shape maintenance. Given that archaea possess the ancestral form of eukaryotic histone, this study has important implications for understanding the evolution of histone function.
Novel Approaches Reveal that Toxoplasma gondii Bradyzoites within Tissue Cysts Are Dynamic and Replicating EntitiesIn Vivo
Authors: Watts Elizabeth , Zhao Yihua , Dhara Animesh , Eller Becca , Patwardhan Abhijit , Sinai Anthony P. 
Published: September 2015

ABSTRACTDespite their critical role in chronic toxoplasmosis, the biology ofToxoplasma gondiibradyzoites is poorly understood. In an attempt to address this gap, we optimized approaches to purify tissue cysts and analyzed the replicative potential of bradyzoites within these cysts. In order to quantify individual bradyzoites within tissue cysts, we have developed imaging software, BradyCount 1.0, that allows the rapid establishment of bradyzoite burdens within imaged optical sections of purified tissue cysts. While in general larger tissue cysts contain more bradyzoites, their relative “occupancy” was typically lower than that of smaller cysts, resulting in a lower packing density. The packing density permits a direct measure of how bradyzoites develop within cysts, allowing for comparisons across progression of the chronic phase. In order to capture bradyzoite endodyogeny, we exploited the differential intensity of TgIMC3, an inner membrane complex protein that intensely labels newly formed/forming daughters within bradyzoites and decays over time in the absence of further division. To our surprise, we were able to capture not only sporadic and asynchronous division but also synchronous replication of all bradyzoites within mature tissue cysts. Furthermore, the time-dependent decay of TgIMC3 intensity was exploited to gain insights into the temporal patterns of bradyzoite replicationin vivo. Despite the fact that bradyzoites are considered replicatively dormant, we find evidence for cyclical, episodic bradyzoite growth within tissue cystsin vivo. These findings directly challenge the prevailing notion of bradyzoites as dormant nonreplicative entities in chronic toxoplasmosis and have implications on our understanding of this enigmatic and clinically important life cycle stage.IMPORTANCEThe protozoanToxoplasma gondiiestablishes a lifelong chronic infection mediated by the bradyzoite form of the parasite within tissue cysts. Technical challenges have limited even the most basic studies on bradyzoites and the tissue cystsin vivo. Bradyzoites, which are viewed as dormant, poorly replicating or nonreplicating entities, were found to be surprisingly active, exhibiting not only the capacity for growth but also previously unrecognized patterns of replication that point to their being considerably more dynamic than previously imagined. These newly revealed properties force us to reexamine the most basic questions regarding bradyzoite biology and the progression of the chronic phase of toxoplasmosis. By developing new tools and approaches to study the chronic phase at the level of bradyzoites, we expose new avenues to tackle both drug development and a better understanding of events that may lead to reactivated symptomatic disease.