bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a suite of
Proteus mirabilis
-derived urea-inducible promoters
Authors: Fitzgerald Madison J. , Scavone Abigail , Moolaveesala Chaitra , Pearson Melanie M. , Mobley Harry L. T. 
Published: October 2024

ABSTRACT

Catheter-associated urinary tract infections (CAUTIs) are a significant burden on healthcare systems, accounting for up to 40% of hospital-acquired infections globally. A prevalent CAUTI pathogen,
Proteus mirabilis,
is an understudied Gram-negative bacterium. One sequela of
P. mirabilis
CAUTI is the production of urinary stones, which complicates treatment and clearing of the infection. Stone formation is induced by the activity of urease, a nickel-metalloenzyme that is regulated by UreR in a urea-dependent manner. As urea is abundant in the urinary tract, urease genes are highly expressed during experimental UTI. We sought to leverage the urease promoter to create an expression system that would enable urea-inducible expression of genes during
in vitro
experiments as well as during experimental UTI. During preliminary studies, we observed unexpectedly high levels of basal expression of the urease promoter. This was somewhat dependent on the presence of regulator UreR. To further develop this expression system, we generated a series of reporter constructs to assess the impact of specific promoter elements on promoter activity in the presence and absence of urea. Elements of interest included known regulatory binding sites, alternative translational start sites, and single-nucleotide polymorphisms identified through comparative genomics. This work describes a suite of urea-inducible promoters, constructed during this study, that exhibit a variety of expression dynamics, providing a customizable platform for gene expression.

IMPORTANCE

Urea is an inexpensive molecule that can easily be supplied during
in vitro
experiments. A urea-inducible promoter would also be activated by environments where urea naturally occurs, such as in the urinary tract. Thus, the development of a urea-inducible system for selective gene expression is of great interest to the field of uropathogenesis as it would enable selective gene induction during experimental urinary tract infection. This expression system would also have important applications for recombinant protein production in biotech and manufacturing.
Quantifying bacterial efflux within subcellular domains of
Pseudomonas aeruginosa
Authors: Li Yujie , Wilhelm Michael J. , Wu Tong , Hu Xiao-Hua , Ruiz Oscar N. , Dai Hai-Lung 
Published: October 2024

ABSTRACT

Molecular efflux is a mechanism through which bacteria actively expel undesirable substances. This is a crucial line of defense against toxic chemicals in harsh environments. Understanding how efflux works is critical for designing antimicrobial strategies. Though much is already known about efflux proteins, important details about the mechanisms of efflux (e.g., importance of specific subcellular domains and ejection rates) have yet to be experimentally quantified. Herein, we use the nonlinear optical technique, second harmonic light scattering, to simultaneously measure the efflux rates from the periplasm and cytosol of a Gram-negative bacterium. The influence of efflux on the uptake kinetics of a mild antimicrobial, malachite green (MG), by
Pseudomonas aeruginosa
was quantified. It is observed that efflux primarily occurs from the periplasm and is two orders of magnitude faster than from the cytosol. Efflux pumps activate to maintain MG concentrations in the periplasm below 1 µM, while efflux from the cytosol maintains MG concentration below 0.1 µM. Efflux pumps are shown to saturate when exogenous MG concentrations are greater than 25 µM, while the cytosol efflux function saturates at >15 µM. Finally, efflux pumps can simultaneously eject different compounds, as proven by experiments with both MG and hexane, a known effluxable compound.

IMPORTANCE
Molecular efflux pumps are a crucial defense mechanism that protects bacteria from an otherwise unchecked influx of toxic molecules present in the extracellular environment. The efflux functions constitute a significant hindrance to antimicrobial efficacy. While much is now known regarding the structure of these channels, knowledge of the influence of efflux in individual subcellular domains and the associated ejection rates is still lacking. Using the nonlinear optical technique, second-harmonic light scattering, we have measured the threshold concentrations for pump activation, saturation concentrations, and efflux rates from both the periplasm and cytosol in living Gram-negative bacteria. The quantified efflux data in the different subcellular compartments not only provide a clear mechanistic understanding but also are critical for developing antimicrobial strategies.
Genomic characterization of a clonal emergent
Salmonella
Minnesota lineage in Brazil reveals the presence of a novel megaplasmid of resistance and virulence
Authors: dos Santos Anamaria M. P. , Panzenhagen Pedro , Ferrari Rafaela G. , Portes Ana Beatriz , de Jesus Ana Carolina , Ochioni Alan , Rodrigues Dália , Toro Magaly , Meng Jianghong , Allard Marc , Conte-Junior Carlos A. 
Published: October 2024

ABSTRACT

Salmonella
Minnesota has emerged in Brazil as the predominant serovar in poultry and poultry products, along with
Salmonella
Heidelberg. To understand the emergence of
Salmonella
Minnesota over the last few years in Brazil, we performed a comparative analysis between 69 selected
S
. Minnesota genomes from Pathogen Detection database and 65 clonal emergent genomes isolated from Brazil. We demonstrate the presence of multidrug resistance genes against tetracycline [
tet
(A)], sulfonamide (
sul2
), and AmpC beta-lactamase (
bla
CMY-2
) in emergent genomes, along with the carriage of a megaplasmid of resistance and virulence (~210 kb), designated pESM (plasmid for emergent
Salmonella
Minnesota). pESM is an IncC/A2 plasmid predicted to increase
S
. Minnesota environmental tolerance to mercury (
mer
operon) and provide resistance to tetracycline and ampicillin due to the presence of
tet
(A) and
bla
CMY-2
, respectively. Moreover, pESM carries the yersiniabactin siderophore (high-pathogenicity island of
Yersinia
) related to the iron uptake. The temporal inference demonstrated that the most recent common ancestor dated from ~1978 and that the clonal emergent genomes carrying the pESM belong to a completely different lineage of
S
. Minnesota. Our results indicate that the presence of pESM likely contributes to the emergence of
S
. Minnesota and is precisely related to the successful spread of this particular clonal lineage in Brazil.

IMPORTANCE

Salmonella
Minnesota has emerged in Brazil as one of the leading serovars related to human and animal infection, presenting high virulence and antibiotic resistance to drugs classified as the highest priority for clinical treatment in humans. This study performed whole-genome sequencing, temporal analysis, and phylogenetics to understand the genetic insights related to the emergence of
Salmonella
Minnesota in Brazil. Long-read sequencing has led to the identification and characterization of a unique megaplasmid carrying virulence, antibiotic resistance, and heavy-metal tolerance genes, which may play a central role in
S
. Minnesota’s successful emergence in Brazil and possibly worldwide. The potentially high transmissibility of this plasmid between clones and serovars represents a risk to public health since its acquisition may increase
Salmonella
’s fitness, virulence, resistance, and persistence. Understanding the genetic aspects related to the emergence of serovars can help devise measures to mitigate the spread of hazardous multidrug-resistant strains.
The efflux system CdfX exports zinc that cannot be transported by ZntA in
Cupriavidus metallidurans
Authors: Schulz Vladislava , Galea Diana , Schleuder Grit , Strohmeyer Philipp , Große Cornelia , Herzberg Martin , Nies Dietrich H. 
Published: October 2024

ABSTRACT

Cupriavidus metallidurans
is able to survive exposure to high concentrations of transition metals, but is also able to grow under metal starvation conditions. A prerequisite of cellular zinc homeostasis is a flow equilibrium combining zinc uptake and efflux processes. The mutant strain ∆e4 of the parental plasmid-free strain AE104 with a deletion of all four chromosomally encoded genes of previously known efflux systems ZntA, CadA, DmeF, and FieF was still able to efflux zinc in a pulse-chase experiment, indicating the existence of a fifth efflux system. The gene
cdfX
, encoding a protein of the cation diffusion facilitator (CDF) family, is located in proximity to the
cadA
gene, encoding a P-type ATPase. Deletion of
cdfX
in the ∆e4 mutant resulted in a further decrease in zinc resistance. Pulse-chase experiments with radioactive
65
Zn(II) and stable-isotope-enriched
67
Zn(II) provided evidence that CdfX was responsible for the residual zinc efflux activity of the mutant strain ∆e4. Reporter gene fusions with
cdfX-lacZ
indicated that the MerR-type regulator ZntR, the main regulator of
zntA
expression, was responsible for zinc- and cadmium-dependent upregulation of
cdfX
expression, especially in mutant cells lacking one or both of the previously characterized efflux systems, ZntA and CadA. Expression of
zntR
also proved to be controlled by ZntR itself as well as by zinc and cadmium availability. These data indicate that the
cdfX-cadA
region provides
C. metallidurans
with a backup system for the zinc-cadmium-exporting P-type ATPase ZntA, with CdfX exporting zinc and CadA cadmium.

IMPORTANCE
Bacteria have evolved the ability to supply the important trace element zinc to zinc-dependent proteins, despite external zinc concentrations varying over a wide range. Zinc homeostasis can be understood as adaptive layering of homeostatic systems, allowing coverage from extreme starvation to extreme resistance. Central to zinc homeostasis is a flow equilibrium of zinc comprising uptake and efflux reactions, which adjusts the cytoplasmic zinc content. This report describes what happens when an imbalance in zinc and cadmium concentrations impairs the central inner-membrane zinc efflux system for zinc by competitive inhibition for this exporter. The problem is solved by activation of Cd-exporting CadA or Zn-exporting CdfX as additional efflux systems.
Detection of
Hepatovirus A
(HAV) in wastewater indicates widespread national distribution and association with socioeconomic indicators of vulnerability
Authors: Zulli Alessandro , Chan Elana M. G. , Boehm Alexandria B. 
Published: October 2024

ABSTRACT

Wastewater-based epidemiology, which seeks to assess disease occurrence in communities through measurements of infectious disease biomarkers in wastewater, may represent a valuable tool for understanding the occurrence of hepatitis A infections in communities. In this study, we measured concentrations of
Hepatovirus A
(HAV) RNA, in samples from 191 wastewater treatment plants spanning 40 US states and the District of Columbia from September 2023 to June 2024 and compared the measurements with traditional measures of disease occurrence. Nationally, 13.76% of the 21,079 wastewater samples were positive for HAV RNA, and both concentrations and positivity rates were associated with NNDSS hepatitis A case data nationally (Kendall rank correlation coefficient = 0.20, concentrations; and 0.33, positivity rate; both
P
< 0.05). We further demonstrated that higher rates of wastewater HAV detection were positively associated with socioeconomic indicators of vulnerability including homelessness and drug overdose deaths (both
P
< 0.0001). Areas with above average levels of homelessness were 48% more likely to have HAV wastewater detections, while areas with above average levels of drug overdose deaths were 14% more likely to have HAV wastewater detections. Using more granular case data, we present a case study in the state of Maine that reinforces these results and suggests a potential lead time for wastewater over clinical case detection and exposure events. The ability to detect HAV RNA in wastewater before clinical cases emerge could allow public health officials to implement targeted interventions like vaccination campaigns.

IMPORTANCE
Despite the existence of a highly effective vaccine for hepatitis A, outbreaks in vulnerable populations remain common. The disease can be asymptomatic or subclinical, and disproportionately impacts populations with inadequate access to healthcare, leading to a severe underestimation of the occurrence of this viral infection. This study investigates the potential for wastewater measurements of biomarkers of the causative agent of hepatitis A (HAV RNA) to provide insights into disease occurrence. Results highlight the potential for wastewater-based epidemiology to be a complementary tool to traditional surveillance for monitoring and controlling HAV transmission.
Effects of global change drivers on the expression of pathogenicity and stress genes in dryland soil fungi
Authors: Romero-Olivares Adriana L. , Lopez Andrea , Catalan-Dibene Jovani , Ferrenberg Scott , Jordan Samuel E. , Osborne Brooke 
Published: October 2024

ABSTRACT

The impacts of global climate change on dryland fungi have been understudied even though fungi are extremely sensitive to changes in the environment. Considering that many fungi are pathogens of plants and animals, including humans, their responses to anthropogenic change could have important implications for public health and food security. In this study, we investigated the potential physiological responses (i.e., metatranscriptomics) of pathogenicity and stress in dryland fungi exposed to global change drivers, drought, and the physical disturbance associated with land use. Specifically, we wanted to assess if there was an increase in the transcription of genes associated to pathogenicity and stress in response to global change drivers. In addition, we wanted to investigate which pathogenicity and stress genes were consistently differentially expressed under the different global change conditions across the heterogeneous landscape (i.e., microsite) of the Chihuahuan desert. We observed increased transcription of pathogenicity and stress genes, with specific genes being most upregulated in response to global change drivers. Additionally, climatic conditions linked to different microsites, such as those found under patches of vegetation, may play a significant role. We provide evidence supporting the idea that environmental stress caused by global change could contribute to an increase of pathogenicity as global climate changes. Specifically, increases in the transcription of stress and virulence genes, coupled with variations in gene expression, could lead to the onset of pathogenicity. Our work underscores the importance of studying dryland fungi exposed to global climate change and increases in existing fungal pathogens, as well as the emergence of new fungal pathogens, and consequences to public health and food security.

IMPORTANCE
The effects of global climate change on dryland fungi and consequences to our society have been understudied despite evidence showing that pathogenic fungi increase in abundance under global climate change. Moreover, there is a growing concern that global climate change will contribute to the emergence of new fungal pathogens. Yet, we do not understand what mechanisms might be driving this increase in virulence and the onset of pathogenicity. In this study, we investigate how fungi respond to global change drivers, physical disturbance, and drought, in a dryland ecosystem in terms of pathogenicity and stress. We find that indeed, under global change drivers, there is an increase in the transcription and expression of genes associated to pathogenicity and stress, but that microclimatic conditions matter. Our study shows the importance of investigating dryland fungi exposed to global climate change and impacts on our society, which may include threats to public health and food security.
Unraveling the role of cAMP signaling in
Giardia
: insights into PKA-mediated regulation of encystation and subcellular interactions
Authors: Shih Han-Wei , Alas Germain C. M. , Paredez Alexander R. 
Published: October 2024

ABSTRACT

cAMP plays an important role as a second messenger in the stage transition of various protozoan parasites. This signaling pathway relies on multiple effectors, such as protein kinase A (PKA), exchange protein activated by cAMP, and cAMP-response element binding protein transcription factors, to initiate signal transduction in humans. The
Giardia
genome only contains two adenylate cyclases (ACs), a single phosphodiesterase (PDE) and a single known PKA effector, and the specific functions of these components are not fully understood. In our previous research, we demonstrated the important role of AC2-dependent cAMP signaling in promoting the encystation program. Using the NanoBit technology, we emphasized the significance of AC2-dependent cAMP biosynthesis in regulating the dissociation of the PKA regulatory domain (PKAr) and PKA catalytic domain (PKAc). In this study, our objectives are twofold: first, we used the newly developed Split-Halo to examine subcellular interactions of
Gl
PKAr and
Gl
PKAc in
Giardia
; and second, we investigated whether PKAc regulates encystation-specific proteins. Our findings revealed distinct subcellular locations where
Gl
PKAr and
Gl
PKAc interacted during the trophozoite stage, including the flagella, basal bodies, and cytoplasm. Upon exposure to encystation stimuli, the interaction shifted from the flagella to the cytosol. Knockdown of
Gl
PKAc resulted in the downregulation of encystation-specific genes, leading to the production of fewer viable and water-resistant cysts indicating a role for PKA in the transcriptional regulation of encystation. These discoveries contribute to a deeper understanding of the cAMP signaling pathway and its important role in governing
Giardia
’s encystation process.

IMPORTANCE
The precise timing of interactions and subcellular compartmentation play crucial roles in signal transduction. The co-immunoprecipitation assay (CO-IP) has long been utilized to validate protein–protein interactions; however, CO-IPs lack spatial and temporal resolutions. Our recent study used the NanoBit assay, which showcased the reversible protein–protein interaction between PKAr and PKAc in response to cAMP analogs and encystation stimuli. Expanding on this groundwork, this study employs the Split-Halo assay to uncover the subcellular compartments where the PKAr and PKAc protein–protein interactions take place and respond to encystation stimuli. Taken together, these molecular tools provide spatiotemporal information on the protein–protein interaction, which will be useful in the field.
Effect of combined probiotics and doxycycline therapy on the gut–skin axis in rosacea
Authors: Yu Jie , Duan Yan , Zhang Meng , Li Qi , Cao Miao , Song Weixin , Zhao Feiyan , Kwok Lai-Yu , Zhang Heping , Li Ruiya , Sun Zhihong 
Published: October 2024

ABSTRACT

Rosacea is a chronic inflammatory skin condition marked by facial erythema, telangiectasia, and acne-like eruptions, affecting millions worldwide. While antibiotics remain a common treatment, prolonged use has significant adverse effects and can lead to antibiotic resistance. This study evaluated the impact of combined probiotics and doxycycline treatment on rosacea, emphasizing the gut–skin axis. Sixty rosacea patients were randomly assigned to the probiotic, placebo, or control groups. After a 2-week doxycycline treatment, participants underwent a 3-month intervention with either a placebo, probiotic, or no further treatment. Clinical outcomes were assessed at baseline and after the 14-week intervention. Our results showed that probiotic administration improved facial skin conditions, alleviated inflammation, and reduced facial skin microbiota diversity while enhancing gut microbiota heterogeneity. Multivariate analysis identified microbial markers distinguishing the probiotic group from the control and placebo groups, and some markers were associated with skin health parameters. After the probiotic intervention, some facial skin-associated taxa, such as
Aquabacterium
sp.,
UBA4096
sp. 1,
UBA4096
sp. 2, and
Yimella indica
, decreased in abundance. Additionally, the fecal microbiota of the probiotic group was enriched in specific gut microbes, including
Streptococcus parasanguinis
,
Erysipelatoclostridium ramosum
, and
Coprobacillus cateniformis
, while showing a reduced abundance of
Bacteroides vulgatus
. These changes were associated with reduced facial sebum levels and a lower physician’s global assessment score. Finally, fewer antibiotic resistance genes, particularly tetracycline resistance genes, were detected in the probiotic group compared with the control and placebo groups. Our study supports the existence of a gut–skin axis and the application of probiotics in managing rosacea.

IMPORTANCE
This research elucidates rosacea management with novel insights into probiotic use alongside doxycycline, showing dual benefits in symptom relief and inflammation reduction in patients. The study maps probiotic-induced shifts in gut and skin microbiota, underscoring microbial shifts correlating with skin health improvements. Crucially, it deciphers the gut–skin axis modulation by probiotics, proposing a method to curb antibiotic resistance in rosacea therapies. This study furnishes robust evidence for probiotics in rosacea, advancing our grasp of the gut–skin relationship.
Translithospheric carbonatite magmatic system of the world’s largest REE deposit, Bayan Obo, China
Authors: Xue Guoqiang , Zhang Ji’en , Chen Weiying , Wu Xin , Wang Jian , Di Qingyun , Xu Ya , Zhao Yonggang , Li Xianhua , Zhao Yulong , Mitchell Ross 
Published: October 2024

Abstract
Critical rare earth elements (REEs) are often sourced from carbonatite worldwide. In some cases, the magmatic system at depth is not well depicted due to a lack of geophysical constraints. The world’s largest REE deposit, Bayan Obo of the North China craton, offers a template for tracing such a system using 2-D electrical resistivity imaging. We first restore modifications triggered by two newly discovered Mesozoic thrust faults. The thrust fault along Boluotou, Dongjielegele, and south West Pit displaced gneiss and the carbonatite, which was intruded by Permian granite, in the hanging wall from Kulue ~30 km south of Bayan Obo, as evidenced by four similar left-step en echelon high aeromagnetic anomalies. The second Haoqin–North Jianshan fault thrust is another carbonatite at Boluotou, East-Main-West pits, and Dongjielegele and Paleoproterozoic metasediments over Paleozoic sediments, that correlate with the Shuiyuantou klippe to constrain a displacement of ~14 km. After restoration, both carbonatites are revealed to be situated above two low resistivity zones. We propose a Mesoproterozoic translithospheric carbonatite magmatic system consisting of a frozen zone of partial melt and mantle sill complexes stemming from as deep as near the lithosphere–asthenosphere boundary at 60–70 km depth and two magma pathways, plumbing magma upward across the crust to generate two carbonatites with multiple branches at the surface. Our geophysical constraint on the magmatic system of this giant REE deposit can serve as a blueprint for understanding and exploring other existing and new REE deposits.