bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bacterial motility depends on a critical flagellum length and energy-optimised assembly
Authors: Halte Manuel , Popp Philipp F. , Hathcock David , Severn John , Fischer Svenja , Goosmann Christian , Ducret Adrien , Charpentier Emmanuelle , Tu Yuhai , Lauga Eric , Erhardt Marc , Renault Thibaud T. 
Published: June 2024

The flagellum is the most complex macromolecular structure known in bacteria and comprised of around two dozen distinct proteins. The main building block of the long, external flagellar filament, flagellin, is secreted through the flagellar type-III secretion system at a remarkable rate of several tens of thousands amino acids per second, significantly surpassing the rates achieved by other pore-based protein secretion systems. The evolutionary implications and potential benefits of this high secretion rate for flagellum assembly and function, however, have remained elusive. In this study, we provide both experimental and theoretical evidence that the flagellar secretion rate has been evolutionarily optimized to facilitate rapid and efficient construction of a functional flagellum. By synchronizing flagellar assembly, we found that a minimal filament length of 2.5 µm was required for swimming motility. Biophysical modelling revealed that this minimal filament length threshold resulted from an elasto-hydrodynamic instability of the whole swimming cell, dependent on the filament length. Furthermore, we developed a stepwise filament labeling method combined with electron microscopy visualization to validate predicted flagellin secretion rates of up to 10,000 amino acids per second. A biophysical model of flagellum growth demonstrates that the observed high flagellin secretion rate efficiently balances filament elongation and energy consumption, thereby enabling motility in the shortest amount of time. Taken together, these insights underscore the evolutionary pressures that have shaped the development and optimization of the flagellum and type-III secretion system, illuminating the intricate interplay between functionality and efficiency in assembly of large macromolecular structures.Significance statementOur study demonstrates how protein secretion of the bacterial flagellum is finely tuned to optimize filament assembly rate and flagellum function while minimizing energy consumption. By measuring flagellar filament lengths and bacterial swimming after initiation of flag-ellum assembly, we were able to establish the minimal filament length necessary for swimming motility, which we rationalized physically as resulting from an elasto-hydrodynamic instability of the swimming cell. Our bio-physical model of flagellum growth further illustrates how the physiological flagellin secretion rate is optimized to maximize filament elongation while conserving energy. These findings illuminate the evolutionary pressures that have shaped the function of the bacterial flagellum and type-III secretion system, driving improvements in bacterial motility and overall fitness.
Assessment of Antimicrobial Therapy in EradicatingChlamydia muridarumin Research Mice: Immune Status and its Impact on Outcomes
Authors: Palillo Michael B , Carrasco Sebastian E , Mishkin Noah , Palillo Jack A , Lynch Denise B , Lawton Samira , Aydin Mert , Mourino Anthony , Lipman Neil S , Ricart Arbona Rodolfo J 
Published: June 2024

AbstractChlamydia muridarum (Cm) is a moderately prevalent, gram-negative, intracellular bacterium that affects laboratory mice, causing subclinical to severe disease, depending on the host’s immune status. The effectiveness of various antibiotic regimens aimed at eradicating Cm in both immunodeficient and immunocompetent laboratory mice was evaluated. NSG mice were cohoused with Cm-shedding BALB/cJ mice for 14 days to simulate natural exposure. Four groups of 8 infected NSG mice were treated for 7 days with either 0.08% sulfamethoxazole and 0.016% trimethoprim (TMS) in water, 0.0625% doxycycline in feed, 0.124%/0.025% TMS in feed, or 0.12% amoxicillin in feed. A control group was provided standard water and feed. The impact of treatment on gastrointestinal microbiota (GM) was performed using next-generation shotgun sequencing on the last day of treatment. TMS and Amoxicillin had negligible effects on GM, while doxycycline had the largest effect. All antibiotic treated NSG mice exhibited clinical disease, including dehydration, hunched posture, >20% weight loss, and dyspnea, leading to euthanasia 21-40 days post-treatment (32.6 ± 4.2 days; mean ± SD). Untreated controls were euthanized 14-33 days post-exposure (23.75 ± 5.9 days). All mice were fecal PCR positive for Cm at euthanasia. Histological evaluation revealed multifocal histiocytic and neutrophilic bronchointerstitial pneumonia and/or bronchiolitis featuring prominent intralesional chlamydial inclusion bodies in all mice. Subsequently, groups of 8 C57BL/6J, BALB/cJ, NOD.SCID, and NSG mice infected with Cm were treated with 0.124%/0.025% TMS in feed for 7 (BALB/cJ and C57BL/6J) or 21 days (NSG and NOD.SCID). All immunocompetent and NOD.SCID mice were negative for Cm by PCR 14 days post-treatment, remained clinically normal and had no evidence of Cm infection at necropsy, all NSG mice remained Cm positive and were euthanized. While these findings highlight the difficulties in eradicating Cm from highly immunodeficient mice, eradication of Cm from immunocompetent or moderately immunocompromised mice with antibiotics is feasible.
Globoside is an essential intracellular factor required for parvovirus B19 endosomal escape in erythroid progenitor cells
Authors: Bieri Jan , Suter Corinne , Caliaro Oliver , Bartetzko Seraina , Bircher Cornelia , Ros Carlos 
Published: June 2024

AbstractHuman parvovirus B19 (B19V), like most parvoviruses, possesses phospholipase A2(PLA2) activity, which is thought to mediate endosomal escape by membrane disruption. Here, we challenge this model and find evidence for a mechanism of B19V entry mediated by the glycosphingolipid globoside without endosome disruption and retrograde transport to the Golgi. We show that B19V PLA2 activity requires specific calcium levels and pH conditions that are not optimal in endosomes. Accordingly, endosomal membrane integrity was maintained during B19V entry. Furthermore, endosomes remained intact when loaded with MS2 bacteriophage particles pseudotyped with multiple B19V PLA2 subunits, providing superior enzymatic potential compared to native B19V. In globoside knockout cells, incoming viruses are arrested in the endosomal compartment and the infection is blocked. Infection can be rescued by promoting endosomal leakage with polyethyleneimine (PEI), demonstrating the essential role of globoside in facilitating endosomal escape. Incoming virus colocalizes with Golgi markers and interfering with Golgi function blocks infection, suggesting that globoside-mediated entry involves the Golgi compartment, which provides conditions favorable for the lipolytic PLA2. Our study challenges the current model of B19V entry and identifies globoside as an essential intracellular receptor required for endosomal escape.
Rhythmic variation in proteomics: challenges and opportunities for statistical power and biomarker identification
Authors: Spick Matt , Isherwood Cheryl M , Gethings Lee , Hassanin Hana , van der Veen Daan R , Skene Debra J. , Johnston Jonathan D 
Published: June 2024

AbstractTime-of-day variation in the molecular profile of biofluids and tissues is a well-described phenomenon, but – especially for proteomics – is rarely considered in terms of the challenges this presents to reproducible biomarker identification. In this work we demonstrate these confounding issues using a small-scale proteomics analysis of male participants in a constant routine protocol following an 8-day laboratory study, in which sleep-wake, light-dark and meal timings were controlled. We provide a case study analysis of circadian and ultradian rhythmicity in proteins in the complement and coagulation cascades, as well as apolipoproteins, and demonstrate that rhythmicity increases the risk of Type II errors due to the reduction in statistical power from increased variance. For the proteins analysed herein we show that to maintain statistical power if chronobiological variation is not controlled for,nshould be increased (by between 9% and 20%); failure to do so would increase β, the chance of Type II error, from a baseline value of 20% to between 22% and 28%. Conversely, controlling for rhythmic time-of-day variation in study design offers the opportunity to improve statistical power and reduce the chances of Type II errors. Indeed, control of time-of-day sampling is a more cost-effective strategy than increasing sample sizes. We recommend that best practice in proteomics study design should account for temporal variation as part of sampling strategy where possible. Where this is impractical, we recommend that additional variance from chronobiological effects be considered in power calculations, that time of sampling be reported as part of study metadata, and that researchers reference any previously identified rhythmicity in biomarkers and pathways of interest. These measures would mitigate against both false and missed discoveries, and improve reproducibility, especially in studies looking at biomarkers, pathways or conditions with a known chronobiological component.
Comprehensive analysis of end-modified long dsDNA donors in CRISPR-mediated endogenous tagging
Authors: Takagi Rioka , Hata Shoji , Tei Chiharu , Mabuchi Akira , Anzai Ryosuke , Fukuyama Masamitsu , Yamamoto Shohei , Chinen Takumi , Toyoda Atsushi , Kitagawa Daiju 
Published: June 2024

AbstractCRISPR-mediated endogenous tagging is a powerful gene editing technique for studying protein dynamics and function in their native cellular environment. While the use of 5’ modified DNA donors has emerged as a promising strategy to improve the typically low efficiency of knock-in gene editing, the underlying mechanisms remain poorly understood. In this study, we conducted a comprehensive analysis of end-modified long linear dsDNA donors in CRISPR-mediated endogenous tagging in human non-transformed cells. In-depth analysis of repair patterns reveals that 5’ biotinylation of dsDNA donors significantly reduces imprecise insertions, thereby enhancing homology-directed repair (HDR)-mediated precise insertion efficiency. Notably, the impact of biotinylation on repair patterns resembles that of non-homologous end joining (NHEJ) pathway inhibition, suggesting its role in preventing NHEJ-mediated mis-integration. Moreover, combining biotin modification with NHEJ inhibitor treatment further improves bi-allelic knock-in efficiency. Overall, this study provides novel insights into the mechanisms by which 5’ modifications enhance precise knock-ins and demonstrates their potential for achieving high-efficient, prercise endogenous tagging in human cells.
Role of Phage–Antibiotic Combinations in Reducing ESBL-Producing and Carbapenem-ResistantEscherichia coli
Authors: Shamsuzzaman Md , Kim Shukho , Kim Jungmin 
Published: June 2024

ABSTRACTThe emergence of extended-spectrumβ-lactamase (ESBL)-producingE. coliand carbapenem-resistantE. coli(CREC) poses a significant global health concern. Here, we isolated and characterized two novel phages and studied their effectiveness with antibiotics against ESBL-producingE. coliand CREC. The isolated phages, EC.W1-9 and EC.W15-4, belonged to thePodoviridaeandMyoviridaefamilies, respectively. They are safe for bacterial control as they do not contain integrase or toxin-coding genes. The phage combination considerably enhanced lytic ability, effectively lysing 61.7% of the 60E. coliisolates, compared to lysis in the 41.6% –55% range by individual phages. Phages EC.W1-9 and EC.W15-4 combined demonstrated 100% susceptibility against differentE. colisequence types, including ST73, ST648, ST2311, ST405, ST7962, ST131, ST13003, and ST167. Additionally, studies showed synergy between antibiotics and phage combinations against ESBL-producingE.coli, with susceptibility of 73.3% and 54% for CREC. The combined treatment of isolated phages and antibiotics significantly increased survival rates in BALB/c mice exposed to various ST types of ESBL-producingE. coliand CREC, including ST131, ST648, and ST410. Survival rates against KBN7288 (ST131) increased by approximately 75% and 50% compared to individual phages EC.W1-9 and EC.W15-4, respectively. When phages and antibiotics were combined, survival rates againstE. coliisolates KBN5617 (ST410), KBN6241 (ST410), and KBN4004 (ST648) ranged from 75% – 100%. Finally, this study highlights the importance of phage and phage-antibiotic combinations to prepare phages for killing different ST types of ESBL-producingE. coliand CREC isolates.IMPORTANCEWhen combined with antibiotics, phage therapy shows promise in fighting multidrug-resistant bacteria. However, antagonism between phages and antibiotics has been reported. This research isolates and characterizes two novel phages, EC.W1-9 and EC.W15-4, from thePodoviridaeandMyoviridaefamilies, respectively, and evaluates their effectiveness against ESBL-producingE. coliand CREC. These phages, lacking integrase or toxin-coding genes, showed significant promise in bacterial control. Combined phage treatment lysed 61.7% ofE.coliisolates, outperforming individual phages. The phage combination showed 100% susceptibility against differentE. colisequence types. Additionally, the synergy between phages and antibiotics increased susceptibility rates to 73.3% for ESBL-producingE. coliand 54% for CREC. In BALB/c mice, combined treatments significantly improved survival rates against variousE. coliisolates. Finally. this study emphasizes the potential of phage and phage-antibiotic combinations in targeting various ST types of ESBL-producingE. coliand CREC.
The Molecules Gateway: a homogeneous, searchable database of 150k annotated molecules from Actinomycetes
Authors: Simone Matteo , Iorio Marianna , Monciardini Paolo , Santini Massimo , Cantù Niccolò , Tocchetti Arianna , Serina Stefania , Brunati Cristina , Vernay Thomas , Gentile Andrea , Aracne Mattia , Cozzi Marco , van der Hooft Justin J.J. , Sosio Margherita , Donadio Stefano , Maffioli Sonia I. 
Published: June 2024

ABSTRACTNatural products are a sustainable resource for drug discovery, but their identification in complex mixtures remains a daunting task. We present an automated pipeline that compares, harmonizes and ranks the annotations of LC-HRMS data by different tools. When applied to 7,400 extracts derived from 6,566 strains belonging to 86 actinomycete genera, it yielded 150,000 molecules after processing over 50 million MS features. The web-based Molecules Gateway provides a highly interactive access to experimental and calculated data for these molecules, along with the metadata related to extracts and producer strains. We show how the Molecules Gateway can be used to rapidly identify known hard to find microbial products, unreported analogs of known families and not yet described metabolites. The Molecules Gateway, which complements available repositories of annotated MS data, is experimentally and computationally homogeneous, and thus amenable to global analyses, which show a large and untapped chemical diversity afforded by actinomycetes.
Bottom-up reconstruction of minimal pyrenoids provides insights into the evolution and mechanisms of carbon concentration by EPYC1 proteins
Authors: Küffner A. M. , Pommerenke B. , Kley L. , Ng J. Z. Y. , Prinz S. , Tinzl-Zechner M. , Claus P. , Paczia N. , Zarzycki J. , Hochberg G. K. A. , Erb T. J. 
Published: June 2024

SummaryMembraneless organelles play essential roles in cellular processes. In various photosynthetic organisms, they confer carbon concentrating mechanisms (CCMs). One example is the pyrenoid inChlamydomonas reinhardtii, a liquid-phase separated organelle that localizes and improves carbon fixation via the intrinsically-disordered protein (IDP) EPYC1. Modern-day pyrenoids are complex structures, which makes it impossible to study the function of EPYC1, especially whether EPYC1 alone confers carbon concentration, and how EPYC1 could have initiated the evolution of pyrenoids. Here, we developed a bottom-up approach to study the function of EPYC1 and its sequence-function space across evolution. We demonstrate that modern-day EPYC1-sequences, but not other IDPs, induce liquid-phase separation of Rubisco into minimal pyrenoids with functional CCMs. Using ancestral sequence reconstruction, we trace the evolution of pyrenoids and demonstrate that selection acted on carboxylation rate, and in selected cases on specificity, providing unexpected perspectives on the design and function of natural and synthetic pyrenoids.
Unlocking the potential ofUvaria chamae: A cream formulation for combattingStaphylococcus aureusskin infections
Authors: Togbé Eskyl , Koudokpon Hornel , Ayéna Aimé Césaire , Assogba Phénix , Hounsa Edna , Watara Amy , Agbodjento Eric , Ohouko Fréjus , Klotoé Jean Robert , Baba-Moussa Lamine , Dougnon Victorien 
Published: June 2024

AbstractStaphylococcus aureus(S. aureus) skin infections remain a prevalent public health concern worldwide, posing ongoing challenges in treatment.Uvaria chamaeroots has demonstrated significant effectiveness againstS. aureus. This study aimed to propose a dermal formulation based onUvaria chamaefor treating bacterial skin infections. The antibacterial activity was first confirmed. Tests including the time-kill test, the erythromycin outer membrane permeability test, and the ATPase/H+ proton pump inhibition test were conducted to explore the antibacterial mode of action. A cream was prepared using the phase inversion technique and subjected to quality control and senso-rheological testing. The cream was tested for antibacterial activity and skin toxicity (OECD guideline 404). Thein vivoantibacterial effect and wound-healing potential of theUvaria chamaecream were then assessed on Wistar rat. The ethanolic extract ofUvaria chamaeroot exhibited bactericidal effects on the testedS. aureusstrains. In combination with erythromycin, the extract enhanced the permeability ofS. aureus’s outer membrane while inhibiting ATPase/H+ proton pumps. The pharmaceutical quality of the produced cream revealed acceptable macroscopic conditions and satisfactory homogeneity. Antibacterial activity evaluation showed significant activity at low concentrations. The cream’s healing properties indicated a significant decrease in wound diameter (p<0.05) over time, culminating in complete healing on day 13 for the treated group, with a retraction percentage of 99.99%. These findings support the safe use ofUvaria chamaeextracts in managingS. aureusskin infections and pave the way for pre-clinical testing and potential market introduction of this cream.
Giant transposons promote strain heterogeneity in a major fungal pathogen
Authors: Gluck-Thaler Emile , Forsythe Adrian , Puerner Charles , Stajich Jason E. , Croll Daniel , Cramer Robert A. , Vogan Aaron A. 
Published: June 2024

AbstractFungal infections are difficult to prevent and treat in large part due to strain heterogeneity. However, the genetic mechanisms driving pathogen variation remain poorly understood. Here, we determined the extent to whichStarships—giant transposons capable of mobilizing numerous fungal genes—generate genetic and phenotypic variability in the human pathogenAspergillus fumigatus. We analyzed 519 diverse strains, including 12 newly sequenced with long-read technology, to reveal 20 distinctStarshipsthat are generating genomic heterogeneity over timescales potentially relevant for experimental reproducibility.Starship-mobilized genes encode diverse functions, including biofilm-related virulence factors and biosynthetic gene clusters, and many are differentially expressed during infection and antifungal exposure in a strain-specific manner. These findings support a new model of fungal evolution whereinStarshipshelp generate variation in gene content and expression among fungal strains. Together, our results demonstrate thatStarshipsare a previously hidden mechanism generating genotypic and, in turn, phenotypic heterogeneity in a major human fungal pathogen.ImportanceNo “one size fits all” option exists for treating fungal infections in large part due to genetic and phenotypic variation among strains. Accounting for strain heterogeneity is thus fundamental for developing efficacious treatments and strategies for safeguarding human health. Here, we report significant progress towards achieving this goal by uncovering a previously hidden mechanism generating heterogeneity in the major human fungal pathogenAspergillus fumigatus: giant transposons calledStarshipsthat span dozens of kilobases and mobilize fungal genes as cargo. By conducting the first systematic investigation of these unusual transposons in a single fungal species, we demonstrate their contributions to population-level variation at the genome, pangenome and transcriptome levels. TheStarshipatlas we developed will not only help account for variation introduced by these elements in laboratory experiments but will serve as a foundational resource for determining howStarshipsshape clinically-relevant phenotypes, such as antifungal resistance and pathogenicity.
Promiscuous NAD-dependent dehydrogenases enable efficient bacterial growth on the PET monomer ethylene glycol
Authors: Ren Minrui , Li Danni , Addison Holly , Noteborn Willem E. M. , Andeweg Els H. , Glatter Timo , de Winde Johannes H. , Rebelein Johannes G. , Lamers Meindert H. , Schada von Borzyskowski Lennart 
Published: June 2024

AbstractEthylene glycol is widely used as antifreeze agent and monomer of the ubiquitous plastic PET (polyethylene terephthalate). Its global production amounts to more than 50 million tons per year, and it constitutes an environmental pollutant of increasing concern. Although it is generally accepted that bacteria oxidize ethylene glycol to use it as growth substrate, the enzymes involved in this process are not well understood. Here we show that the soil bacteriumParacoccus denitrificansis able to assimilate ethylene glycol efficiently via NAD-dependent alcohol and aldehyde dehydrogenases. Using comparative proteome analysis, we identify a previously unknown gene cluster that is strongly expressed in the presence of ethylene glycol. We report the kinetic parameters and cryo-EM structures of EtgB and EtgA, the key enzymes encoded by thisetggene cluster. These novel biocatalysts pave the way for more efficient biotechnological conversion of ethylene glycol. We furthermore show that the transcriptional activator EtgR controls expression of theetggene cluster. Directed evolution ofP. denitrificanson ethylene glycol results in faster growing strains, which is enabled by increased activities of EtgB and EtgA. Bioinformatic analysis reveals that theetggene cluster and variants thereof are widely distributed among Proteobacteria, suggesting a previously underappreciated role of NAD-dependent dehydrogenases in microbial ethylene glycol assimilation.