bluuebunny/crossref_metadata_2025 huggingface dataset:
Higher-fold topological excitations in phononic and electronic phases of chiral-type BaXY (X = Pt, Pd; Y= P, As, Sb, Bi) materials: A first-principles investigation
Authors: Paul Bibhash , P. C. Sreeparvathy , Villaos Rovi Angelo B. , Huang Zhi-Quan , Lin Hsin , Chuang Feng-Chuan
Published: February 2025

Non-symmorphic chiral crystals hosting higher-fold fermions and bosons are of great interest due to the recent experimental realizations. The specialty of these higher-fold fermions and bosons is that they possess long Fermi arcs connecting the Brillouin zone boundaries. In this work, using first-principles calculations, we examine the higher-fold topological phases in the phonon spectrum and electronic band structure of ternary chiral crystals BaXY (X = Pt, Pd; Y = P, As, Sb, Bi). Our detailed analysis reveals the symmetry-protected spin-1 Weyl and charge-2 Dirac topological phonon phase in the phonon spectrum. Contrastingly, the electronic states exhibit intriguing fourfold and sixfold fermionic states under spin–orbit coupling. Interestingly, we found the emergence of giant topological surface Fermi arcs in both phonon and electronic phases. Our findings highlight the coexistence of higher-fold topological phases in both the electronic and phononic states in all of the studied ternary chiral crystals, which may serve future topological device applications.
Features of Successful Talent-Development Environments in Norwegian Professional Women’s Football
Authors: Sæther Stig Arve , Banne Henriette Bolstad , Larsen Carsten Hvid , Storm Louise Kamuk , Schlawe Anusofia , Henriksen Kristoffer
Published: February 2025

The aim of this study was to examine the talent development of a junior-elite team in a women’s football club in Norway. Using a holistic ecological approach, this study was designed as a single exemplary case study of a club in a preclub merge phase caused by a potential merger between a women-only club, FC Dragons and a men-only club, FC Spiders (both clubs are pseudonyms). The data collection included interviews, observations, and document analysis. We found the women’s junior-elite team, FC Dragons, to be characterized by similar characteristics as the men’s club, such as support of sporting goals by the wider environment, a focus on long-term development with little focus on results, training groups with supportive relationships, a coherent organizational culture, and integration of efforts. The women’s team environment was furthermore characterized by access to proximal role models (i.e., the senior players) and closer relationships between coaches of the club’s junior and senior teams. Based on the findings, we discuss how the context of women’s football impacted the talent development environment given to the players in the club.
A sequencing-based screening method identifies regulators of EGFR signaling from nonviable mutants in
Caenorhabditis elegans
Authors: Schwartz Hillel T. , Sternberg Paul W.
Published: February 2025

Suppressor screens can identify genetic modifiers of biochemical pathways but generally require that the suppressed mutant be viable and fertile. We developed a screening method that obviated this requirement and enabled the identification of mutations that partially suppressed the early developmental arrest and lethality caused by loss of the epidermal growth factor (EGF) receptor ortholog LET-23 in
Caenorhabditis elegans
. We chemically mutagenized animals carrying the loss-of-function allele
let-23
(
sy15
), recovered
let-23
(
sy15
) homozygotes that escaped early developmental arrest but were nevertheless inviable, and sequenced their genomes. Testing of candidate causal mutations identified 11 genes that, when mutated, mitigated the early lethality caused by loss of EGF signaling. These included genes encoding homologs of the small guanosine triphosphatase (GTPase) Ras (
let-60
), which is a downstream effector of LET-23, and of regulators of the small GTPase Rho, including the homolog of the phosphotyrosine-binding protein TENSIN (
tns-1
). We also recovered suppressing mutations in genes encoding nuclear proteins that protect against DNA damage, including the homolog of MutS homolog 4 (
him-14
). Genetic experiments were consistent with the repression of Rho activity or the activation of the DNA damage response compensating for the loss of EGF signaling. This sequencing-based, whole-animal screening method may be adapted to other organisms to enable the identification of mutations for which the phenotype does not allow the recovery of viable animals.
Dulling the sweet tooth
Authors: VanHook Annalisa M.
Published: February 2025

A gut microbe that is reduced in diabetes produces a metabolite that dulls the appetite for sugar.
The impact of ibezapolstat and other
Clostridioides difficile
infection-relevant antibiotics on the microbiome of humanized mice
Authors: Wolfe Trenton M. , Jo Jinhee , Pinkham Nick V. , Garey Kevin W. , Walk Seth T.
Published: February 2025

Ibezapolstat (IBZ) is a competitive inhibitor of the bacterial Pol IIIC enzyme in clinical development for the treatment of
Clostridioides difficile
infection (CDI). Previous studies demonstrated that IBZ carries a favorable microbiome diversity profile compared to vancomycin (VAN). However, head-to-head comparisons with other CDI antibiotics have not been done. The purpose of this study was to compare microbiome changes associated with IBZ to other clinically used CDI antibiotics. Groups of germ-free (GF) mice received a fecal microbiota transplant from one of two healthy human donors and were subsequently exposed to either IBZ, VAN, fidaxomicin (FDX), metronidazole (MET), or no antibiotic (control). 16S rRNA encoding gene sequencing of temporally collected stool samples was used to compare the gut microbiome perturbations between treatment and no-drug control groups. Among the tested antibiotics, the most significant change in microbiome diversity was observed in MET-treated mice. Each antibiotic had a unique effect, but changes in alpha and beta diversities following FDX- and IBZ-treated groups were less pronounced than those observed in VAN- or MET-treated groups. By the end of therapy, both IBZ and FDZ increased the relative abundance of
Bacteroidota
(phylum), with IBZ additionally increasing the relative abundance of
Actinomycetota
(phylum). In microbiome-humanized mice, IBZ and FDX had smaller effects on gut microbiome diversity than VAN and MET. Notable differences were observed between the microbiome of IBZ- and FDX-treated groups, which may allow for differentiation of these two antibiotics in future studies.
Minimal changes in microbial abundances and diversity over 7 years of emplacement for modules of compacted bentonite exposed to natural groundwater
Authors: Sidhu Harmanpreet S. , Engel Katja , Ford Sian E. , Keech Peter , Behazin Mehran , Binns W. Jeffrey , Srikanthan Nivetha , Simpson Myrna J. , Neufeld Josh D. , Slater Gregory F.
Published: February 2025

The multi-national
in situ
Materials Corrosion Test (MaCoTe), being conducted at the Grimsel Test Site in Switzerland, assesses the stability of bentonite as it may be utilized within deep geological repositories (DGRs), which are proposed for the safe, long-term disposal of used nuclear fuel. This experiment provides an opportunity for long-term assessments of changes in microbial communities associated with compacted subsurface bentonite samples exposed to a natural groundwater. Leveraging samples from MaCoTe, herein, we report temporal data for the abundance and community composition of microorganisms associated with compacted bentonite samples emplaced over 7 years under
in situ
subsurface conditions. Phospholipid fatty acid analysis, 16S rRNA gene quantification and sequencing, cultivation, and natural organic matter analyses all indicated no significant changes for microbial community abundances associated with inner layers of bentonite samples over the 7 years. While microbial abundances did not change in the inner layers, the PLFA data suggest potential changes in microbial community composition and could also indicate prolonged microbial turnover rates. Overall, the results support microbial stability in compacted bentonite exposed to DGR-like conditions for at least 7 years.

IMPORTANCE

Long-term assessments of changes in microbial activity in compacted low-biomass bentonite systems analogous to deep geological repositories (DGRs) are critical to test conditions for stable engineered bentonite barrier components. This study assesses long-term temporal changes in microbial communities of compacted bentonites exposed to natural groundwater. It offers 7-year data that indicate stability of bentonite-based materials intended for use in engineered barrier systems of a DGR for the safe, long-term disposal of used nuclear fuel, with wider implications for microbial persistence in a deep subsurface environment.
Current prevalence and therapeutic strategies for porcine
Streptococcus suis
in China
Authors: Lv Ruoyi , Zhang Wenjing , Sun Zhigang , Si Xiaohui , Dong Hong , Liu Xiaoye
Published: February 2025

Porcine
Streptococcus suis
is a zoonotic bacterial pathogen that poses serious threats to both human and animal health.
S. suis
is ubiquitously transmitted from the swine industry to the environments and human communities. However, the ambiguous epidemiological patterns and the escalating risk of antimicrobial resistance render
S. suis
infections a considerable challenge. Here, we review the current prevalence of
S. suis
infection worldwide, including identified bacterial strains, routes of infection, and transformation of resistance genes. This comprehensive overview of the prevalent patterns in
S. suis
offers detailed insights into therapeutic approaches for porcine infections and alternative strategies to address emerging resistant strains, highlighting potential multitarget prevention and treatment options to combat
S. suis
infection.
A strain of
Streptococcus mitis
inhibits biofilm formation of caries pathogens via abundant hydrogen peroxide production
Authors: Williams Isabella , Tuckerman Jacob S. , Peters Daniel I. , Bangs Madisen , Williams Emily , Shin Iris J. , Kaspar Justin R.
Published: February 2025

Commensal oral streptococci that colonize supragingival biofilms deploy mechanisms to combat competitors within their niche. Here, we determined that
Streptococcus mitis
more effectively inhibited biofilm formation of
Streptococcus mutans
compared to other oral streptococci. This phenotype was common among all isolates of
S. mutans
, but was specific to a single strain of
S. mitis
, ATCC 49456. We documented ATCC 49456 to accumulate four to five times more hydrogen peroxide (H
2
O
2
) than other
Streptococcus
species tested, and 5–18 times more than other
S. mitis
strains assayed.
S. mutans
biofilm formation inhibition was dependent on cell contact/proximity and reduced when grown in media containing catalase or with a
S. mitis
mutant of pyruvate oxidase (
spxB; pox
), confirming that SpxB-dependent H
2
O
2
production was a major antagonistic factor. Addition of
S. mitis
within hours after
S. mutans
inoculation was effective at reducing biofilm biomass, but not for 24 h pre-formed biofilms in an SpxB-dependent manner. Transcriptome analysis revealed responses for both
S. mitis
and
S. mutans
, with several
S. mutans
differentially expressed genes following a gene expression pattern we have previously described, while others being unique to the interaction with
S. mitis
. Finally, we show that
S. mitis
also affected coculture biofilm formation of several other commensal streptococci as well as cariogenic
Streptococcus sobrinus
. Our study shows that strains with abundant H
2
O
2
production are effective at inhibiting initial growth of caries pathogens like
S. mutans
, but are less effective at disrupting pre-formed biofilms and have the potential to influence the stability of other oral commensal strains.

IMPORTANCE

Antagonistic properties displayed by oral bacteria have been sought as therapeutic approaches against dental caries pathogens like
Streptococcus mutans
. An emergent theme has been the ability of select strains that produce high amounts of hydrogen peroxide to effectively inhibit the growth of
S. mutans
within
in vitro
and
in vivo
models. Our study builds on these previous findings by determining that
Streptococcus mitis
ATCC 49456 is a high hydrogen peroxide producer, compared to other
Streptococcus
species as well as additional strains of
S. mitis
. In addition to
S. mutans
, we show that ATCC 49456 also affects biofilm formation of other oral streptococci, a non-desirable trait that should be weighed heavily for strains under consideration as probiotics. Further phenotypic characterization of strains like
S. mitis
ATCC 49456 in mixed-species settings will allow us to hone in on qualities that are optimal for probiotic strains that are intended to prevent the emergence of odontopathogens.
Single-cell delineation of strain-specific HIV-1 Vif activities using dual reporter sensor cells and live cell imaging
Authors: Guerrero Jorge F. , Zimdars Laraine L. , Bruce James W. , Becker Jordan T. , Evans Edward L. , Torabi Soroosh , Striker Rob , Berry Scott M. , Sherer Nathan M.
Published: February 2025

Human immunodeficiency virus type 1 (HIV-1) genome diversification is a key determinant of viral evolution and the pathogenesis of HIV/AIDS. Antiretroviral therapy is non-curative, and in the context of monitoring the latent reservoir, precision tools are needed to detect and enumerate HIV-1 genomes as well as to assess their heterogeneity, replication potential, and predict responses to therapy. Current sequencing-based methodologies are often unable to confirm intact genomes and most cell-based reporters provide limited information pertaining to viral fitness. In this study, we describe dual reporter sensor cells (DRSCs), an imaging-based reporter system designed to detect HIV-1 infection and measure several independent attributes of the virus in a single-cell high-content assay. We show that the DRSC assay can be used to measure infection, viral gene activation kinetics, and quantify viral circumvention of host antiviral responses. Using the DRSCs, we confirmed markedly different functional heterogeneity for
vif
alleles derived from diverse HIV-1 strains and subtypes affecting both rates of APOBEC3G degradation and the cell cycle. Furthermore, the assay allowed for the delineation of virus co-receptor preference (X4- vs R5-tropism) and visualization of virion assembly. Overall, our study illustrates proof-of-principle for a multivariate imaging-based cell-based system capable of detecting HIV-1 and studying viral genetic variability with greater data richness relative to prior available modalities.

IMPORTANCE

Human immunodeficiency virus type 1 (HIV-1) is highly heterogeneous and constantly mutating. These changes drive immune evasion and can cause treatment efforts to fail. Here, we describe the “dual reporter sensor cell” (DRSC) assay; a novel imaging-based approach that allows for the detection of HIV-1 infection coupled with a multivariate definition of several independent phenotypic aspects of viral genome activity in a single integrated assay. We validate the DRSC system by studying lab-adapted and patient isolate-derived versions of the viral Vif accessory protein, confirming marked differences in the capacity of diverse
vif
alleles to mediate downregulation of antiviral APOBEC3G proteins and dysregulate the cell cycle.
Quorum sensing employs a dual regulatory mechanism to repress T3SS gene expression
Authors: Paul Payel , Podicheti Ram , Geyman Logan J. , Baker Elizabeth N. , Papenfort Kai , Rusch Douglas B. , van Kessel Julia C.
Published: February 2025

The type III secretion system (T3SS) is a needle-like complex used by numerous bacterial pathogens in host infection to inject exotoxins into the host cell cytoplasm. The T3SS is a known virulence factor in the shrimp pathogen
Vibrio campbellii
. The ~40 genes comprising the
V. campbellii
T3SS are regulated by a network of transcription factors in response to changes in the cell’s environment: cell density (quorum sensing; QS), temperature, calcium, and host cell contact. Under positive environmental stimuli, the master T3SS transcription factor ExsA activates the expression of the four structural T3SS operons required for needle formation. Previous studies identified a key role of the master QS transcription factor LuxR: repression of
exsA
transcription via DNA binding at the
exsBA
promoter. Here, we uncovered a new regulatory role of LuxR: post-translational repression of ExsA activity via transcriptional repression of the gene encoding the anti-anti-activator ExsC. In
V. campbellii
, ExsC is a positive regulator of T3SS transcription; deletion of
exsC
decreases ExsA-dependent transcription activation of the T3SS structural promoters. Through genetic epistasis and
in vitro
biochemical assays, we show that LuxR directly binds the
exsC
promoter upstream of ExsA and represses transcription of
exsC
. Our findings collectively show that
V. campbellii
responds to high cell density signals to shut down ExsA-dependent expression of the T3SS via two mechanisms. We postulate that this dual regulatory mechanism by LuxR enables both the rapid inactivation of existing ExsA protein and blocks its further synthesis, leading to a rapid shutdown of T3SS activity at high cell density.

IMPORTANCE

Vibrio campbellii
utilizes the type III secretion system (T3SS) as a mechanism of pathogenesis, which is a highly studied “injectisome” complex that delivers exotoxins into host cells during infection. The T3SS pathogenicity island in
V. campbellii
comprises ~40 genes that are organized into four structural operons. In this study, we determined that quorum sensing—a method of bacterial communication—regulates T3SS genes both at the transcriptional and post-translational levels to shut down T3SS gene expression at high population densities.
Fission yeast metabolome dynamics during phosphate starvation and replenishment
Authors: Sanchez Ana M. , Kyaw Aye K. , Violante Sara Nunes , Garg Angad , Cross Justin R. , Shuman Stewart
Published: February 2025

Inorganic phosphate is an essential nutrient acquired by cells from their environment and assimilated into myriad intracellular metabolites and macromolecules. Here, we characterize the metabolic responses of fission yeast to a 24 h interval of phosphate starvation, during which cells enter a state of G0 quiescence. Time-resolved profiling revealed that many key phosphometabolites were progressively depleted, including (i) NTPs, NDPs, and dNTPs; (ii) coenzyme A, NAD
+
, NADP
+
, NADH, and ADP-ribose; (iii) glycolysis pathway intermediates upstream of pyruvate; (iv) pentose phosphate pathway intermediates from 6-phosphogluconate to sedoheptulose-7-phosphate; (v) nucleotide sugars GDP-hexose, UDP-glucose/galactose, and UDP-GalNAc/GlcNAc; and (vi) phospholipid precursors glycerol-3-phosphate, CDP-choline, and glycerophosphocholine. By contrast, early Krebs cycle intermediates accumulated during phosphate starvation. Other metabolic changes included the following: (i) interdiction of
de novo
pyrimidine synthesis; (ii) depletion of
S
-adenosylmethionine and
S
-adenosylhomocysteine; (iii) transient accumulation of polyamine biosynthetic intermediates putrescine and 5-methylthioadenosine; (iv) accumulation of betaine (correlating with an increase in expression of
atd1
mRNA encoding aldehyde dehydrogenase); and (v) depletion of aminoadipate pathway intermediates 2-oxoadipate, 2-aminoadipate, and saccharopine. Replenishing phosphate after 24 h of starvation resulted in restoration of the pre-starvation metabolome (over 2 to 12 h) as cells exited quiescence and resumed growth.

IMPORTANCE

Fission yeast
Schizosaccharomyces pombe
is a valuable model system to study cellular phosphate homeostasis and the adaptive responses to chronic phosphate starvation. Previous analyses focused on changes in the fission yeast transcriptome and proteome during phosphate starvation-induced durable G0 quiescence. Here, we deployed metabolomics to survey the scope and temporal order of metabolite changes during 24 h of phosphate starvation and the kinetics of metabolic recovery after cells starved for 24 h are replenished with phosphate. These results contribute to a multi-omics understanding of how phosphate status impacts cell cycle, gene expression, metabolism, and chronological lifespan.
The HOPS and vCLAMP protein Vam6 connects polyphosphate with mitochondrial function and oxidative stress resistance in
Cryptococcus neoformans
Authors: Sánchez-León Eddy , Bhalla Kabir , Hu Guanggan , Lee Christopher W. J. , Lagace Melissa , Jung Won Hee , Kronstad James W.
Published: February 2025

Cryptococcus neoformans
is considered one of the most dangerous fungal threats to human health, and the World Health Organization recently ranked it in the critical priority group for perceived public health importance. Proliferation of
C. neoformans
within mammalian hosts is supported by its ability to overcome nutritional limitations and endure stress conditions induced by the host immune response. Previously, we reported that the Vam6/Vps39/TRAP1-domain protein Vam6 was crucial for vacuolar morphology, iron acquisition, and virulence. However, the molecular mechanisms underlying the pleiotropic phenotypes resulting from loss of Vam6 remain poorly understood. In this study, we determined that Vam6 has roles in the HOPS complex for endomembrane trafficking to the vacuole and in the vCLAMP membrane contact site between the vacuole and mitochondria. Importantly, both of these roles regulate polyphosphate (polyP) metabolism, as demonstrated by a defect in trafficking of the VTC complex subunit Vtc2 for polyphosphate synthesis and by an influence on mitochondrial functions. In the latter case, Vam6 was required for polyP accumulation in response to electron transport chain inhibition and for overcoming oxidative stress. Overall, this work establishes connections between endomembrane trafficking, mitochondrial functions, and polyP homeostasis in
C. neoformans
.

IMPORTANCE

A detailed understanding of stress resistance by fungal pathogens of humans may provide new opportunities to improve antifungal therapy and combat life-threatening diseases. Here, we used a
vam6
deletion mutant to investigate the role of the homotypic fusion and vacuole protein sorting (HOPS) complex in mitochondrial functions and polyphosphate homeostasis in
Cryptococcus neoformans
, an important fungal pathogen of immunocompromised people including those suffering from HIV/AIDS. Specifically, we made use of mutants defective in late endocytic trafficking steps to establish connections to oxidative stress and membrane trafficking with mitochondria. In particular, we found that mutants lacking the Vam6 protein had altered mitochondrial function, and that the mutants were perturbed for additional mitochondria and vacuole-related phenotypes (e.g., membrane composition, polyphosphate accumulation, and drug sensitivity). Overall, our study establishes connections between endomembrane trafficking components, mitochondrial functions, and polyphosphate homeostasis in an important fungal pathogen of humans.
A proteomic and functional view of intrabacterial lipid inclusion biogenesis in mycobacteria
Authors: Dargham Tonia , Aguilera-Correa John Jairo , Avellan Romain , Mallick Ivy , Celik Léa , Santucci Pierre , Brasseur Gael , Poncin Isabelle , Point Vanessa , Audebert Stéphane , Camoin Luc , Daher Wassim , Cavalier Jean-François , Kremer Laurent , Canaan Stéphane
Published: February 2025

During infection and granuloma formation, pathogenic mycobacteria store triacylglycerol as intrabacterial lipid inclusions (ILIs). This accumulation of nutrients provides a carbon source for bacterial persistence and slows down intracellular metabolism.
Mycobacterium abscessus
(
Mab
), a rapidly growing non-tuberculous actinobacterium, produces ILI throughout its infection cycle. Here,
Mab
was used as a model organism to identify proteins associated with ILI accumulation on a global scale. By using the APEX2 proximity labeling method in an
in vitro
model for ILI accumulation, we identified 228 proteins possibly implicated in ILI biosynthesis. Fluorescence microscopy of strains overexpressing eight ILI-associated proteins (IAP) candidates fused to superfolder green fluorescent protein showed co-localization with ILI. Genetic inactivation of these potential IAP-encoding genes and subsequent lipid analysis emphasized the importance of MAB_3486 and MAB_4532c as key enzymes influencing triacylglycerol storage. This study underscores the dynamic process of ILI biogenesis and advances our understanding of lipid metabolism in pathogenic mycobacteria. Identifying major IAP in lipid accumulation offers new therapeutic perspectives to control the growth and persistence of pathogenic mycobacteria.

IMPORTANCE

This study sheds light into the complex process of intracellular lipid accumulation and storage in the survival and persistence of pathogenic mycobacteria, which is of clinical relevance. By identifying the proteins involved in the formation of intrabacterial lipid inclusions and revealing their impact on lipid metabolism, our data may lead to the development of new therapeutic strategies to target and control pathogenic mycobacteria, potentially improving outcomes for patients with mycobacterial infections.
Blanking on blanks: few insect microbiota studies control for contaminants
Authors: Williamson Elisabeth M. , Hammer Tobin J. , Hogendoorn Katja , Eisenhofer Raphael
Published: February 2025

Research on insect-microbe relationships is booming, with DNA sequencing being the most commonly used method to describe insect microbiota. However, sequencing is vulnerable to contamination, especially when the sample has low microbial biomass. Such low-biomass samples are common across insect taxa, developmental stages, and tissue types. Identifying putative contaminants is essential to distinguish between true microbiota and introduced contaminant DNA. It is therefore important that studies control for contamination, but how often this is done is unknown. To investigate the status quo of contamination control, we undertook a systematic literature review to quantify the prevalence of negative control usage and contamination control across the literature on insect microbiota (specifically bacterial communities) over a 10 year period. Two-thirds of the 243 insect microbiota studies evaluated had not included blanks (negative controls), and only 13.6% of the studies sequenced these blanks and controlled for contamination in their samples. Our findings highlight a major lack of contamination control in the field of insect microbiota research. This result suggests that a number of microbes reported in the literature may be contaminants as opposed to insect-associated microbiota and that more rigorous contamination control is needed to improve research reliability, validity, and reproducibility. Based on our findings, we recommend the previously developed guidelines outlined in the RIDE checklist, with the addition of one more guideline. We refer to this as the RIDES checklist, which stands for Report methodology, Include negative controls, Determine the level of contamination, Explore contamination downstream, and State the amount of off-target amplification.

IMPORTANCE

Our systematic review reveals a major lack of methodological rigor within the field of research on insect-associated microbiota. The small percentage of studies that control for contamination suggests that an unknown but potentially considerable number of bacteria reported in the literature could be contaminants. The implication of this finding is that true microbiota may be masked or misrepresented, especially in insects with low microbial biomass.
Inter-protomer opening cooperativity of envelope trimers positively correlates with HIV-1 entry stoichiometry
Authors: Katte Revansiddha H. , Xu Wang , Han Yang , Hong Xinyu , Lu Maolin
Published: February 2025

HIV-1 entry to host cells is fulfilled by fusion, mediated by surface glycoprotein envelope (Env) trimers upon interaction with host receptors. The entry stoichiometry (T) defining the number of Env trimers required for fusion remains elusive. Stoichiometry was previously estimated using mathematical modeling of infectivity curves of pseudoviruses surface-decorated with heterotrimers containing wild-type and entry-deficient Env. Nevertheless, previous models rarely co-considered inter-protomer opening cooperativity (S, reflecting how CD4-induced conformational changes in one protomer affect the opening of adjacent protomers) and virion trimer number distributions, while experiments were limited to pseudoviruses. Here, we factored these two parameters into our models and included replication-competent virions. We provided simultaneous estimates of T and S under varying trimer number distributions and offered 2D stoichiometry maps for different Env strains. Our results depicted the interplay between viral infectivity and stoichiometry tuned by the number of trimers per virion. The estimates for all tested Env strains were prevalently higher (T ≥ 7 for BG505 or JR-FL, T ≥ 13 for NL4-3) than reported. A high degree of inter-protomer opening cooperativity was observed for the neutralization-sensitive NL4-3, while neutralization-resistant BG505 and JR-FL showed a low to intermediate degree. Entry stoichiometry and opening cooperativity were strikingly positive-correlated, implying tied inter-protomer and inter-Env cooperative interactions. Our findings provided an in-depth view of Env cooperativities during HIV-1 entry.

IMPORTANCE

The sparsely distributed envelope (Env) trimers on the surface of HIV-1 work collaboratively to mediate viral entry into the host, the early step of infection. The number of interacting trimers with host receptors required for entry awaits elucidation. Here, we explored the cooperative interplay among and within Env trimers, shedding light on a previously overlooked dimension of HIV-1 entry. For the first time, we presented distributions of estimated parameters depicting the number of Env trimers and degrees of inter-protomer opening cooperativities using biologically relevant mathematic models combined with virion infectivity measurements. Our results demonstrated that the quantity of required functional trimers positively correlates with inter-protomer opening cooperativity, a feature conserved across various strains. Our findings underscore cooperative behavior as an inherent characteristic of Env dynamics during HIV-1 entry. These insights enhance our understanding of HIV-1 infection mechanisms and could inform strategies for developing effective inhibitors or neutralizing agents.
Heparanase, a host gene that potently restricts retrovirus transcription
Authors: Waxman Brandon , Salka Kyle , Timilsina Uddhav , Umthong Supawadee , Shukla Deepak , Stavrou Spyridon
Published: February 2025

Heparanase (HPSE) is a heterodimeric β-D-glucuronidase that is critical in mammalian cells for the enzymatic cleavage of membrane-associated heparan sulfate moieties. Apart from its enzymatic function, HPSE has important non-enzymatic functions, which include transcriptional regulation, chromatin modification, and modulation of various signaling pathways. Interestingly, while
HPSE
is an interferon-stimulated gene, past reports have shown that it has proviral properties for many different viruses, including herpes simplex virus 1, as it assists virus release from infected cells. However, as of yet, no antiviral functions associated with HPSE have been described. Here, we show that HPSE utilizes a hitherto unknown mechanism to restrict retroviruses by targeting the step of proviral transcription. Moreover, we demonstrate that HPSE blocks transcription initiation by targeting the SP1 transcription factor. Finally, we illustrate that the antiretroviral effect of HPSE is independent of its enzymatic activity. This report describes a novel antiviral mechanism utilized by HPSE to inhibit retrovirus infection.

IMPORTANCE

Heparanase (HPSE) has emerged as an important factor that has proviral functions for a number of viruses, including herpes simplex virus and hepatitis C virus, by assisting in virus egress. However, HPSE is an interferon-stimulated gene and, thus, is a part of the host antiviral defense. Nothing is known about the antiviral functions of HPSE. Here, we examine in depth the role of HPSE during retrovirus infection using two retroviruses, human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus. In this report, we show that mouse, but not human, HPSE blocks retrovirus infection by targeting provirus transcription. HPSE sequesters the SP1 transcription factor away from the proviral promoter, thereby inhibiting transcription initiation. In conclusion, our findings identify a novel antiviral function of HPSE and its potential role as an inhibitor of zoonotic transmission of retroviruses.
Campylobacter jejuni
resistance to human milk involves the acyl carrier protein AcpP
Authors: Zhou Bibi , Garber Jolene M. , Butcher James , Muszynski Artur , Casey Rebekah L. , Huynh Steven , Archer-hartmann Stephanie , Porfírio Sara , Rogers Ashley M. , Azadi Parastoo , Parker Craig T. , Ng Kenneth K. S. , Hines Kelly M. , Stintzi Alain , Szymanski Christine M.
Published: February 2025

Campylobacter jejuni
is a common foodborne pathogen worldwide that is associated with high rates of morbidity and mortality among infants in low- to middle-income countries (LMICs). Human milk provides infants with an important source of nutrients and contains antimicrobial components for protection against infection. However, recent studies, including our own, have found significantly higher levels of
Campylobacter
in diarrheal stool samples collected from breastfed infants compared to non-breastfed infants in LMICs. We hypothesized that
C. jejuni
has unique strategies to resist the antimicrobial properties of human milk. Transcriptional profiling found human milk exposure induces genes associated with ribosomal function, iron acquisition, and amino acid utilization in
C. jejuni
strains 81–176 and 11168. However, unidentified proteinaceous components of human milk prevent bacterial growth. Evolving both
C. jejuni
isolates to survive in human milk resulted in mutations in genes encoding the acyl carrier protein (AcpP) and the major outer membrane porin (PorA). Introduction of the PorA/AcpP amino acid changes into the parental backgrounds followed by electron microscopy showed distinct membrane architectures, and the AcpP changes not only significantly improved growth in human milk, but also yielded cells surrounded with outer membrane vesicles. Analyses of the phospholipid and lipooligosaccharide (LOS) compositions suggest an imbalance in acyl chain distributions. For strain 11168, these changes protect both evolved and 11168∆
acpP
G33R
strains from bacteriophage infection and polymyxin killing. Taken together, this study provides insights into how
C. jejuni
may evolve to resist the bactericidal activity of human milk and flourish in the hostile environment of the gastrointestinal tract.

IMPORTANCE

In this study, we evolved
C. jejuni
strains which can grow in the presence of human milk and found that cell membrane alterations may be involved in resistance to the antimicrobial properties of human milk. These bacterial membrane changes are predominantly linked to amino acid substitutions within the acyl carrier protein, AcpP, although other bacterial components, including PorA, are likely involved. This study provides some insights into possible strategies for
C. jejuni
survival and propagation in the gastrointestinal tract of breastfed infants.
Investigation of the
Cyanothece
nitrogenase cluster in
Synechocystis
: a blueprint for engineering nitrogen-fixing photoautotrophs
Authors: Liu Deng , Bandyopadhyay Anindita , Liberton Michelle , Pakrasi Himadri B. , Bhattacharyya-Pakrasi Maitrayee
Published: February 2025

The nitrogenase gene cluster of unicellular diazotrophic cyanobacteria, such as
Cyanothece
, is frequently selected by nature for nitrogen-fixing partnerships with eukaryotic phototrophs. The essential cluster components that confer an advantage in such partnerships remain underexplored. To use this cluster for the development of synthetic, phototrophic nitrogen-fixing systems, a thorough and systematic analysis of its constituent genes is necessary. An initial effort to assess the possibility of engineering this cluster into non-diazotrophic phototrophs led to the generation of a
Synechocystis
6803 strain with significant nitrogenase activity. In the current study, a refactoring approach was taken to determine the dispensability of the non-structural genes in the cluster and define a minimal gene set for constructing a functional nitrogenase for phototrophs. Using a bottom-up strategy, the
nif
genes from
Cyanothece
51142 were re-organized to form new operons. The genes were then seamlessly removed to determine their essentiality in the nitrogen fixation process. We demonstrate that besides the structural genes
nifHDK
,
nifBSUENPVZTXW
, as well as
hesAB
, are important for optimal nitrogenase function in a phototroph. We also show that optimal expression of these genes is crucial for efficient nitrogenase activity. Our findings provide a solid foundation for generating synthetic systems that will facilitate solar-powered conversion of atmospheric nitrogen into nitrogen-rich compounds, a stride toward a greener world.

IMPORTANCE

Integrating nitrogen fixation genes into various photosynthetic organisms is an exciting strategy for converting atmospheric nitrogen into nitrogen-rich products in a green and energy-efficient way. In order to facilitate this process, it is essential that we understand the fundamentals of the functioning of a prokaryotic nitrogen-fixing machinery in a non-diazotrophic, photoautotrophic cell. This study examines a nitrogenase gene cluster that has been naturally selected on multiple occasions for a nitrogen-fixing partnership by eukaryotic photoautotrophs and provides a basic blueprint for designing a photosynthetic organism with nitrogen-fixing ability.
CsrA-mediated regulation of a virulence switch in
Acinetobacter baumannii
Authors: Singh Raja , Pérez-Varela María , Colquhoun Jennifer M. , Kröger Carsten , Hamrock Fergal J. , Shaibah Ali , Neidle Ellen L. , Rather Philip N.
Published: February 2025

CsrA is an RNA binding protein that functions as a global regulator in bacteria. We demonstrate that, in
Acinetobacter baumannii
, CsrA acts as a positive regulator of the switch from virulent (VIR-O) to avirulent (AV-T) subpopulations. This regulation is mediated, in part, by CsrA interfering with Rho-dependent termination in the mRNA leader region of the
ABUW_1645
gene, encoding the primary TetR-type transcriptional regulator that drives cells from the VIR-O to the AV-T state. We demonstrate that CsrA directly binds to the
ABUW_1645
mRNA leader region and interferes with Rho binding. We identify three small RNAs (sRNAs) designated CsrB, CsrC, and CsrD that bind to CsrA and inhibit its activity. Individual overexpression of each sRNA greatly decreased the rate of VIR-O to AV-T switching. Individual deletions of each sRNA increased the frequency of VIR-O to AV-T switching, with loss of CsrB giving the highest increase at 2.4-fold. The expression of each sRNA was strongly dependent on the GacA response regulator and the expression of each sRNA was higher in VIR-O cells than in AV-T variants. This regulation provides a mechanism for the differential control of CsrA activity between VIR-O and AV-T variants.

IMPORTANCE

The World Health Organization has ranked
Acinetobacter baumannii
atop its “priority pathogens” list highlighting the urgent need for new therapeutics against this pathogen. Many
A. baumannii
strains including AB5075 can rapidly switch between cell subpopulations that are virulent or avirulent. In this study, we identify the RNA binding protein CsrA as an important regulator of this switch. Since this switch represents an “Achilles Heel” for pathogenesis, our work may shed light on new mechanisms to lock cells into the avirulent state to block disease.
Draft genomes of six methicillin-resistant
Staphylococcus aureus
in a Philippine tertiary hospital with insights on putative antimicrobial and phage resistance mechanisms
Authors: Carascal Mark B. , Gonzales Jo-Eliz M. , Tañedo Ma. Mona Joy T. , Papa Donna May D. , Destura Raul V. , Henson Karl Evans R.
Published: February 2025

The genomes of six methicillin-resistant
Staphylococcus aureus
(MRSA) revealed CC5(ST5)-SCC
mec
-IVc-
spa
-t105-PVL
+
(
n
= 3), CC5(ST6)-SCC
mec
-IVc-
spa
-t10002 (
n
= 1), CC5(ST5)-SCC
mec
-IVa-
spa
-t002 (
n
= 1), and CC8(ST8)-SCC
mec
-IVa-
spa
-t008-PVL
+
(
n
= 1) genotypes. All possessed multiple antibiotic resistance genes (putative
mecA
and
blaZ,
and
dfrG
for cotrimoxazole-resistant strains), intact staphylococcal prophages, and putative antiphage systems. The results could aid in MRSA infection management and control.
Complete genome sequence of
Veillonella parvula
strain PK1910
Authors: Naumann Allison A. , Siddiqui Danyal , Kerns Kristopher A. , Hendrickson Erik L. , Kotsakis Georgios A. , McLean Jeffrey S.
Published: February 2025

Here, we announce the complete genome sequence of
Veillonella parvula
strain PK1910, obtained from a frozen stock. The genome is composed of one closed contig with a length of 2,213,486 bp, resulting in 98.0× coverage containing 1,979 protein-coding genes, with a GC content of 38.74%.
Metagenome-assembled genomes of acetogenic enrichment obtained from deep subsurface archean granitoids of Koyna Seismogenic Zone, Koyna, India
Authors: Sahu Rajendra Prasad , Sar Pinaki
Published: February 2025

We report 14 metagenome-assembled genomes (MAGs) of acetogenic bacteria (acetogens) enriched from deep (1,679–2,912 m below surface), hot (55°C–74 °C) granitoids of the Koyna Seismogenic Zone, India. These MAGs include
Thermoanaerobacter pseudethanolicus, Exiguobacterium alkaliphilum, Moorella humiferrea, Caldanaerobacter subterraneus
, etc. The study allowed access to the genomes of deep biosphere acetogens.
Draft genome sequence of
Solimonas
sp. strain SE-A11 isolated from the rhizosphere of feral
Brassica napus
in South Korea
Authors: Kim Jihoon , Nam Kyong-Hee , Lee Jun-Woo , Chun Seong-Jun
Published: February 2025

The draft genome of
Solimonas
sp. strain SE-A11 bacterium isolated from the rhizosphere of feral
Brassica napus
in South Korea, represents the first isolate of the genus
Solimonas
obtained from a plant rhizosphere. The strain has a genome size of 4,937,440  bp, a G + C content of 66.5%, and 281 predicted subsystems.
Whole genome sequences of eight
Erwinia amylovora
phages isolated from South Korea
Authors: Roh Eunjung , Duffy Madison E. , Ewool Leslie M. , Grose Julianne H.
Published: February 2025

The gram-negative
Enterobacteriaceae Erwinia amylovora
is the causative agent of fire blight. Herein, we announce the full genome sequencing and annotation of eight
E. amylovora
bacteriophages from apple and pear orchards in South Korea, which have remarkable similarity to
Erwinia
phages previously isolated from the Americas.
Complete genome of the mutualistic symbiont “
Candidatus
Nardonella sp.” Pin-AIST from the black hard weevil
Pachyrhynchus infernalis
Authors: Mizutani Masaki , Moriyama Minoru , Fukatsu Takema , Kakizawa Shigeyuki
Published: February 2025

The complete genome, 226,287 bps in size, of “
Candidatus
Nardonella sp.” Pin-AIST, an obligatory bacterial endosymbiont of the black hard weevil
Pachyrhynchus infernalis
, was sequenced. The extremely reduced endosymbiont genome is specialized for tyrosine synthesis, which contributes to the hardness of the beetle’s exoskeleton.
Near-complete genome of medusavirus euryale, putative third species of the genus
Medusavirus
, isolated from the TaeHwaGang river, Ulsan, South Korea
Authors: Bae Jiwan , Takemura Masaharu
Published: February 2025

We report the isolation and near-complete genome sequencing of medusavirus euryale, a putative third species of the genus
Medusavirus
in the family
Mamonoviridae
, from the TaeHwaGang river in Ulsan, South Korea. Medusavirus euryale has a genome size of approximately 369 kb and contains 446 open reading frames, including two tRNA genes.
Complete genome sequence of
Microbacterium
sp. strain Clip185
Authors: Mitra Mautusi , Stanescu Ana
Published: February 2025

A xenobiotic-degrader and heavy metal-tolerant
Microbacterium sp. strain Clip185
was isolated from a contaminated Tris-Acetate-Phosphate medium culture plate of a green micro-alga,
Chlamydomonas reinhardtii
. Here, we report the complete genome sequence of this strain to provide insights into its metabolic potential and nearest taxonomic neighbor.
In vitro
assays for clinical isolates of sequence type 131
Escherichia coli
do not recapitulate
in vivo
infectivity using a murine model of urinary tract infection
Authors: Rudick Courtney P. , Cox Rachel S. , Bourret Travis J. , Hanson Nancy D.
Published: February 2025

Sequence type 131 isolates are a major cause of cystitis and pyelonephritis. Many studies rely solely on
in vitro
assays to screen for bacterial virulence factors associated with the pathogenicity of clinical isolates of
Escherichia coli
. Few studies have compared
in vitro
findings with
in vivo
infectivity of clinical isolates. The purpose of this study was to evaluate the correlation between
in vitro
assays with the ability to cause cystitis and pyelonephritis in a murine model of urinary tract infection.
In vitro
assays were conducted according to the published protocols and included motility assays, biofilm formation, epithelial cell adhesion and invasion, and curli production. Twenty-one uropathogenic
E. coli
(UPEC) isolates of
E. coli
ST131 and non-ST131 were used for both
in vivo
and
in vitro
studies. Six mice per isolate were inoculated via urethral catheterization. Colony forming units (CFUs) were determined from bladder and kidneys.
In vitro
and
in vivo
correlations were evaluated by multiple linear regression analysis. Pairwise linear regressions showed trendlines with weak positive correlations for motility, adhesion, and invasion and weak negative correlations for hemagglutination, biofilm, and curli production. The ability of
E. coli
ST131 and non-ST131 clinical isolates to cause cystitis and pyelonephritis varied among strains. The
R
2
Pearson correlation value was less than ±0.5 for any pair, indicating little to no statistical association between
in vitro
and
in vivo
findings. These data show that
in vitro
data are not predictive of the ability of ST131
E. coli
to infect and/or cause disease in a mouse model.

IMPORTANCE

Urinary tract infections (UTIs) affect 150 million people annually, and
E. coli
ST131, a pandemic clone, has become responsible for a significant portion of those UTIs. How ST131
E. coli
has become such a successful strain remains to be elucidated. When evaluating bacterial pathogenicity, it is customary to use
in vitro
assays to predict isolate virulence and fitness due to lower cost and ease of experimentation compared with
in vivo
models. It is common to use model organisms like pathogenic
E. coli
CFT073 or a non-pathogenic K12 lab strain as representatives for the entire species. However, our research has shown that model organisms differ from ST131
E. coli,
and
in vitro
assays are poor predictors of ST131 isolate infectivity in a murine model of UTI. As such, research into the mechanisms of fitness/pathogenesis for ST131 infectivity needs to focus on these organisms rather than other types of UPEC.