bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pyruvate kinase directly generates GTP in glycolysis, supporting growth and contributing to guanosine toxicity
Authors: She Fukang , Liu Kuanqing , Anderson Brent W. , Pisithkul Tippapha , Li Yanxiu , Fung Danny K. , McCue Tyler , Mulhern William , Amador-Noguez Daniel , Wang Jue D. 
Published: February 2025

ABSTRACT

Guanosine triphosphate (GTP) is essential for macromolecular biosynthesis, and its intracellular levels are tightly regulated in bacteria. Loss of the alarmone (p)ppGpp disrupts GTP regulation in
Bacillus subtilis
, causing cell death in the presence of exogenous guanosine and underscoring the critical importance of GTP homeostasis. To investigate the basis of guanosine toxicity, we performed a genetic selection for spontaneous mutations that suppress this effect, uncovering an unexpected link between GTP synthesis and glycolysis. In particular, we identified suppressor mutations in
pyk
, which encodes pyruvate kinase, a glycolytic enzyme. Metabolomic analysis revealed that inactivating pyruvate kinase prevents guanosine toxicity by reducing GTP levels. Although traditionally associated with ATP generation via substrate-level phosphorylation,
B. subtilis
pyruvate kinase
in vitro
was found to produce GTP and UTP approximately 10 and three times more efficiently than ATP, respectively. This efficient GTP/UTP synthesis extends to
Enterococcus faecalis
and
Listeria monocytogenes
, challenging the conventional understanding of pyruvate kinase’s primary role in ATP production. These findings support a model in which glycolysis directly contributes to GTP synthesis, fueling energy-demanding processes, such as protein translation. Finally, we observed a synergistic essentiality of the Δ
ndk
Δ
pyk
double mutant specifically on glucose, indicating that pyruvate kinase and nucleoside diphosphate kinase are the major contributors to nucleoside triphosphate production and complement each other during glycolysis. Our work highlights the critical role of nucleotide selectivity in pyruvate kinase and its broader implications in cellular physiology.

IMPORTANCE

In this study, we reveal that pyruvate kinase, a key glycolytic enzyme, primarily generates GTP from GDP in
Bacillus subtilis
, relative to other nucleotide triphosphates, such as ATP. This finding, uncovered through genetic selection for mutants that suppress toxic GTP overaccumulation, challenges the conventional understanding that pyruvate kinase predominantly produces ATP via substrate-level phosphorylation. The substantial role of GTP production by pyruvate kinase suggests a model where glycolysis rapidly and directly supplies GTP as the energy currency to power high GTP-demanding processes such as protein synthesis. Our results underscore the importance of nucleotide selectivity (ATP vs GTP vs UTP) in shaping the physiological state and fate of the cell, prompting further exploration into the mechanisms and broader implications of this selective nucleotide synthesis.
Carbon source, cell density, and the microbial community control inhibition of
V. cholerae
surface colonization by environmental nitrate
Authors: James Jamaurie , Santos Renato E.R.S. , Watnick Paula I. 
Published: February 2025

ABSTRACT

The intestinal diarrheal pathogen
Vibrio cholerae
colonizes the host terminal ileum, a microaerophilic, glucose-poor, nitrate-rich environment. In this environment,
V. cholerae
respires nitrate and increases transport and utilization of alternative carbon sources via the cAMP receptor protein (CRP), a transcription factor that is active during glucose scarcity. Here, we show that
V. cholerae
nitrate respiration in aerated cultures is under control of CRP and, therefore, glucose availability.
V. cholerae
nitrate respiration results in extracellular accumulation of nitrite because
V. cholerae
does not possess the machinery for nitrite reduction. This nitrite inhibits
V. cholerae
biofilm formation via an as-yet unelucidated mechanism that depends on the high cell density master regulator HapR. The genome of
Paracoccus aminovorans
, an intestinal microbe identified in the microbiome of cholera patients that has been shown to enhance
V. cholerae
biofilm accumulation in the neonatal mouse gut, encodes enzymes that reduce nitrite to nitrogen gas. We report that, in nitrate-supplemented co-cultures,
P. aminovorans
metabolizes the nitrite generated by
V. cholerae
and, thereby, enhances
V. cholerae
surface accumulation. We propose that
V. cholerae
biofilm formation in the host intestine is limited by nitrite production but can be rescued by intestinal microbes such as
P. aminovorans
that have the capacity to metabolize nitrite. Such microbes increase
V. cholerae
colonization of the host ileum and predispose to symptomatic infection.

IMPORTANCE

Vibrio cholerae
colonizes the terminal ileum where both oxygen and nitrate are available as terminal electron acceptors.
V. cholerae
biofilm formation is inhibited by nitrate due to its conversion to nitrite during
V. cholerae
respiration. When co-cultured with a microbe that can further reduce nitrite,
V. cholerae
surface accumulation in the presence of nitrate is rescued. The contribution of biofilm formation to ileal colonization depends on the composition of the microbiota. We propose that the intestinal microbiota predisposes mammalian hosts to cholera by consuming the nitrite generated by
V. cholerae
in the terminal ileum. Differences in the intestinal abundance of nitrite-reducing microbes may partially explain the differential susceptibility of humans to cholera and the resistance of non-human mammalian models to intestinal colonization with
V. cholerae
.
Exploring the differential localization of protein kinase A isoforms in
Candida albicans
Authors: Hossain Saif , Liu Zhongle , Robbins Nicole , Cowen Leah E. 
Published: February 2025

ABSTRACT

The cAMP-dependent protein kinase A (PKA) plays important roles in a wide range of biological processes in eukaryotic organisms. In the fungal pathogen
Candida albicans
, PKA is a critical regulator of morphological transitions, which are a key virulence trait. PKA is composed of two catalytic isoforms, Tpk1 and Tpk2, which are often thought to act together in a complex with the regulatory subunit Bcy1. Although Tpk1 and Tpk2 have some redundant functions, they also have distinct cellular functions for which the mechanistic underpinnings remain largely elusive. Here, we constructed functional GFP-tagged fusion proteins for Tpk1, Tpk2, and Bcy1 to explore the localization of PKA isoforms. We observed that the PKA holoenzyme is mainly found in the cytoplasm, as Bcy1 is always excluded from the nucleus. Under glucose-replete conditions, both Tpk1 and Tpk2 translocate into the nucleus from the cytosol. In the presence of glycerol, Tpk1 resides in the cytosol, whereas Tpk2 and Bcy1 become enriched on the vacuolar membrane. As the C-terminal domains of Tpk are highly homologous, we investigated the localization and function of hybrid Tpk proteins with exchanged N-terminal domains. We found the catalytic C-terminus of Tpk1 is required for morphogenesis in solid medium, whereas the C-terminus of Tpk2 is critical for filamentation in liquid. Interestingly, the N-terminus of Tpk2 drives its localization to the vacuolar membrane. Our work highlights environmentally contingent localization patterns for the PKA subunits and suggests that the nuclear localization of Tpk is not sufficient to induce the filamentation program in a leading fungal pathogen of humans.

IMPORTANCE

Fungal pathogens have a devastating impact on human health worldwide. They infect billions of people and kill more than 2.5 million per year.
Candida albicans
is a leading human fungal pathogen responsible for causing life-threatening systemic disease in immunocompromised individuals. A key virulence trait in
C. albicans
is the ability to switch between yeast and filamentous forms. The conserved protein kinase A (PKA) regulates diverse functions in the cell, including growth and filamentation. Although PKA has been studied in
C. albicans
for decades, the subcellular localization of PKA has not been thoroughly investigated. Here, we constructed functional GFP-tagged PKA subunits to explore their localization. We identified differential localization patterns for the PKA subunits that are carbon-source dependent and report that these proteins localize into foci in response to diverse environmental stresses. These findings further our understanding of a critical regulator of growth and virulence in
C. albicans
.
Performance of ddPCR-GNB for microbial diagnosis of suspected bloodstream infection due to the four most common gram-negative bacteria: a prospective, multicenter study
Authors: Weng Shan-shan , Lin Ling , Xie Jian-feng , Hu Bang-chuan , Ma Xue-qing , Xia Jiang , Jiang Yan , Zhou Hua , wu Xiao-yan , Jin Yu-hong , Wu Guo-qiu , Yang Yi , Sun Ren-Hua , Yu Yun-song , Zhao Dong-dong 
Published: February 2025

ABSTRACT

We aimed to validate the performance of ddPCR-GNB, a plasma droplet digital PCR panel targeting the four most common gram-negative bacteria, for patients with suspected bloodstream infection (BSI). Patients suspected of having BSIs were prospectively enrolled. The results of blood culture and ddPCR-GNB were compared, and cases with discordant results were arbitrated on the basis of additional microbiological results and clinical evidence. A total of 1,041 patients were enrolled. Blood culture and ddPCR-GNB results were positive for targeted bacteria in 6.3% and 31.7% of patients, respectively. The overall per-patient sensitivity and specificity of ddPCR-GNB for proven BSIs were 98.5% (95% CI, 91.9% to 99.9%) and 72.8% (95% CI, 69.9% to 75.5%), respectively; the negative predictive value was 99.9% (95% CI, 99.2% to 100%). The discordant results included 265 cases (25.5%) with negative companion blood culture results but positive ddPCR-GNB results and one case vice versa. A total of 23.7% of the cases were attributed to probable (
n
= 126) or possible (
n
= 121) BSIs. If both probable and possible BSIs were assumed to be true positives, the per-patient specificity of ddPCR-GNB would be 97.5%. The ddPCR-GNB panel demonstrated excellent microbial diagnostic performance in identifying targeted bacteria for patients with suspected BSI.

IMPORTANCE
This is the first multicentral study to validate the clinical performance of ddPCR in etiological diagnosis of bloodstream infection. The results showed that ddPCR has high sensitivity and increased detection rate compared with blood culture. The study proved the potential of the ddPCR method in microbial diagnoses.
Synergistic impact of macrolide resistance and H3N2 infection on
M. pneumoniae
outbreak in children
Authors: Chen Jiali , Wang Yingdan , Cheng Juan , Ma Yunping , Zhang Xin , Bai Xuezhou , Rehati Palizhati , Cui Huashun , Wu Fan , Pan Qiuhui , Huang Jinghe 
Published: February 2025

ABSTRACT

In November 2023, there was a substantial increase in the incidence of
Mycoplasma pneumoniae
infections in China following waves of SARS-CoV-2 Omicron variant and influenza outbreaks. This study aimed to elucidate the epidemiological features and clinical implications of
M. pneumoniae
infections in children and explore the potential influence of SARS-CoV-2 Omicron variants and influenza A infections on the
M. pneumoniae
outbreak. Among 38,668 children with lower respiratory tract infections from January to December 2023, 11,919 tested positive for
M. pneumoniae
, predominantly between October and December. The majority of the children with
M. pneumoniae
were aged 5–10 years, with type 1 strains and macrolide-resistant
M. pneumoniae
strains having the highest prevalence rates. Statistical analysis revealed elevated C-reactive protein, neutrophil, and monocyte levels and decreased lymphocyte, basophil, and eosinophil counts in
M. pneumoniae
-positive children.
M. pneumoniae
-positive children also presented significantly increased neutralizing antibody levels against preceding influenza A (H3N2) but not against SARS-CoV-2 Omicron variants. A parallel trend was observed between
M. pneumoniae
and H3N2 prevalence from June to December 2023. The emergence of macrolide-resistant strains and prior influenza A (H3N2) epidemics notably contributed to the
M. pneumoniae
outbreak. These findings suggested that H3N2 infection facilitates
M. pneumoniae
infection through various mechanisms. This study underscores the complex interactions between respiratory pathogens and highlights the need for comprehensive surveillance and response strategies.

IMPORTANCE

This study identified key factors contributing to an outbreak of
Mycoplasma pneumoniae
that affected 11,919 children. The influencing factors included a high prevalence of macrolide-resistant epidemic strains (94.2%) and significantly higher H3N2 neutralizing antibody levels (
P
< 0.0001) stimulated by the preceding H3N2 influenza epidemic. These findings highlight the complex relationship between the prevalence of
M. pneumoniae
and H3N2 infection in children, indicating that it is necessary to consider pathogen interactions in respiratory disease management by continuously monitoring respiratory pathogens. The emergence of macrolide-resistant strains in China and the previous H3N2 influenza epidemic significantly exacerbated the severity of the
M. pneumoniae
outbreak. H3N2 infection potentially amplifies Mycoplasma transmission. This study elucidates the epidemiological and clinical aspects of
M. pneumoniae
infections in children, yields insights regarding the cause of the outbreak, and provides guidance for improving respiratory infection management.
Comparison of
Streptococcus pneumoniae
isolates occurring in optochin-susceptible and optochin-resistant variants by analyzing whole-genome sequencing data
Authors: Vohrnová Sandra , Kozáková Jana , Honskus Michal 
Published: February 2025

ABSTRACT

The paper presents the study of a set of isolates of
Streptococcus pneumoniae
, which comprised two heterogeneous subpopulations, one of which was susceptible and the other resistant to optochin. The aim of the study was to compare the results of serotyping, multilocus sequence typing (MLST), ribosomal multilocus sequence typing (rMLST), and variation analysis of these subpopulations and to investigate the genetic probable causes of optochin resistance. The strains studied were cultured from samples taken from patients with invasive pneumococcal disease in the Czech Republic in 2019 and 2020. A total of 10 studied pairs of isolates were subject to serotyping and whole-genome sequencing (WGS). None of the typing methods (serotyping, MLST, or rMLST) applied to pairs of optochin-susceptible and optochin-resistant isolates revealed differences in serotype, sequence type, or ribosomal sequence type. The WGS data analysis identified point mutations in ATP (adenosine triphosphate) synthase genes in 8 of the 10 optochin-resistant isolates. In seven optochin-resistant isolates, the mutation was found in the
atp
C gene and in one isolate in the
atp
A gene. One of the mutations in the
atp
C gene has not yet been published in the literature; it is a mutation at position 143T > C with an amino acid change of Val48Ala. In 8 out of the 10 optochin-resistant isolates, the possible genetic basis for resistance was identified, involving point mutations in the
atp
A and
atp
C genes. In the remaining two isolates, no clear genetic explanation for the optochin resistance in
S. pneumoniae
was found, based on current knowledge.

IMPORTANCE

Globally, among the most fundamental tests used for the identification of
Streptococcus pneumoniae
isolates is determining susceptibility to optochin. In the last 2 decades, optochin-resistant strains have been frequently reported in the literature, which can lead to the misidentification of
S. pneumoniae
. This study compares whole-genome sequencing data of optochin-susceptible and optochin-resistant subpopulations of
S. pneumoniae
isolates and investigates the genetic probable causes of resistance in the genomes of optochin-resistant subpopulations.
Prospective evaluation of real-world performance and clinical impact of the Biofire FilmArray joint infection panel
Authors: Berinson Benjamin , Tanida Konstantin , Spenke Laura , Krivec Lukas , Keller Johannes , Rolvien Tim , Christner Martin , Lütgehetmann Marc , Aepfelbacher Martin , Klatte Till Orla , Rohde Holger 
Published: February 2025

ABSTRACT

Limitations of culture-based diagnostic approaches in pathogen detection in joint infections (JI) can be overcome by amplification-based, molecular assays. Recently, a syndromic panel PCR (spPCR) assay (Biofire JI panel; BJA) was approved for pathogen identification from synovial fluid (SF). Here, the performance and the clinical impact of the BJA were assessed in comparison to standard of care diagnostics in a prospective cohort of patients presenting with symptoms consistent with JI. One hundred sixty-five synovial fluids underwent analysis using the BJA. The results were compared with culture-based diagnostics. Discrepant results were re-analyzed using species-specific PCRs or 16S-rDNA sequencing. Clinical data from patients were collected to evaluate the impact on patient management. Twenty-seven of 165 (16.3%) synovial fluid cultures grew bacterial pathogens. In 24/27 cases, the BJA results were concordant. In one case, the cultured pathogen was missed, but three additional pathogens were identified. In 11 culture-negative cases, BJA identified a pathogen. Mean turnaround time in culture-positive samples was 14:11 h and 35:17 h in BJA and culture, respectively. In 11 cases, antibiotic therapy was optimized, based on BJA results. This study demonstrates high sensitivity and specificity (96.3% and 97.8%, respectively) of BJA, as well as a shorter turnaround time than culture-based techniques (21 h faster). Based on analysis of clinical data, antibiotic therapy was optimized due to BJA results in 11 cases. Care must be taken, as important pathogens in prosthetic JI are not included in the panel, restricting its value here.

IMPORTANCE
Pathogen detection is critical for targeted management of joint infections; however, cultural detection of pathogens can be challenging. The Biofire Joint Infection Assay (BJA) is a syndromic panel PCR test that allows culture-independent detection of 31 pathogens. The diagnostic performance and clinical impact were evaluated in a cohort of 160 patients with native and prosthetic joint infections. BJA detected concordant pathogens in 24 of 27 culture-positive cases and enabled the detection of additional pathogens in 11 patients. The time to result was significantly shorter than with standard culture-based diagnostics (14 vs 35 h), and BJA allowed optimization of therapy in 11 patients. The data show that BJA is a relevant addition to the diagnostic options for joint infections. Limitations result from incomplete detection of relevant pathogens, especially in prosthetic joint infections. The use of BJA in daily practice must therefore be accompanied by diagnostic stewardship measures.
Simplifying SARS-CoV-2 wastewater-based surveillance using an automated FDA EUA assay
Authors: Sathyanarayana Shivaprasad H. , Robins Ashlee A. , Toledo Diana M. , Gallagher Torrey L. , Tsongalis Gregory J. , Hubbard Jacqueline A. , Lefferts Joel A. , Martin Isabella W. 
Published: February 2025

ABSTRACT

Wastewater-based surveillance (WBS) can track the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in communities. Laboratory methods for this testing involve labor-intensive, multi-step processes. This study assessed the feasibility of performing WBS with an off-label use of an automated commercial SARS-CoV-2 assay that had received Emergency Use Authorization for human diagnostic testing from the United States Food and Drug Administration (FDA EUA). Twenty-four-hour composite samples of primary influent wastewater from seven municipalities in New Hampshire and Vermont were collected between September 2020 and February 2021, and were centrifuged upon receipt. An aliquot of fresh supernatant was immediately tested with the Abbott
m
2000 RealTi
m
e SARS-CoV-2 assay (Abbott Molecular, Des Plaines, IL, USA). Corresponding aliquots were then stored at −80°C until they were thawed, polyethylene glycol (PEG) concentrated, and tested by two PCR-based laboratory-developed tests (LDTs). Wastewater samples (103) were tested with successful detection of SARS-CoV-2 viral RNA by all three methods. Bland-Altman analysis showed overall concordant results with a bias of −0.13 and −0.42 log copies/mL detected by the FDA EUA assay compared to the LDTs. Specimen stability assessment demonstrated a decrease of 33.9% measurable viral RNA after three freeze-thaw cycles. SARS-CoV-2 detection in wastewater using an FDA EUA assay on an automated commercial testing platform performed comparably but with more efficient workflow when compared to two LDTs. This sample-to-answer automated method could save time and labor for surveillance testing, but further validation of its ability to quantitate SARS-CoV-2 viral RNA is necessary.

IMPORTANCE
This proof-of-principle study evaluates an off-label use of an automated United States Food and Drug Administration (FDA) Emergency Use Authorization (EUA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) human diagnostic assay for wastewater surveillance. Compared to standard, labor-intensive, multi-step methods currently in use for wastewater surveillance testing, an off-label use of an FDA EUA assay on an automated platform offers a sample-to-answer testing requiring less labor and a faster turnaround time.