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ELife Jun 2024Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we...
Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also observe functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.
Topics: SARS-CoV-2; Coronavirus Nucleocapsid Proteins; Mutation; COVID-19; Humans; Intrinsically Disordered Proteins; Phosphoproteins; Nucleocapsid Proteins; Thermodynamics; Protein Stability
PubMed: 38941236
DOI: 10.7554/eLife.94836 -
ELife Jun 2024A new study reveals how naturally occurring mutations affect the biophysical properties of nucleocapsid proteins in SARS-CoV-2.
A new study reveals how naturally occurring mutations affect the biophysical properties of nucleocapsid proteins in SARS-CoV-2.
Topics: SARS-CoV-2; Mutation; COVID-19; Humans; Coronavirus Nucleocapsid Proteins; Phosphoproteins
PubMed: 38941233
DOI: 10.7554/eLife.99991 -
Metabolomics : Official Journal of the... Jun 2024Exploring metabolic changes within host E. coli through an untargeted metabolomic study of T7L variants overexpression to optimize engineered endolysins for...
INTRODUCTION
Exploring metabolic changes within host E. coli through an untargeted metabolomic study of T7L variants overexpression to optimize engineered endolysins for clinical/therapeutic use.
AIM AND OBJECTIVE
This study aims to assess the impact of overexpressing T7L variants on the metabolic profiles of E. coli. The two variants considered include T7L-H37A, which has enhanced lytic activity compared to its wild-type protein, and T7L-H48K, a dead mutant with no significant activity.
METHODS
H NMR-based metabolomics was employed to compare the metabolic profiles of E. coli cells overexpressing T7L wild-type protein and its variants.
RESULTS
Overexpression of the T7L wild-type (T7L-WT) protein and its variants (T7L-H48K and T7L-H37A) was compared to RNAP overexpression in E. coli cells using H NMR-based metabolomics, analyzing a total of 75 annotated metabolites, including organic acids, amino acids, sugars, and nucleic acids. The results showed distinct clustering patterns for the two T7L variant groups compared with the WT, in which the dead mutant (H48K) group showed clustering close to that of RNAP. Pathway impact analysis revealed different effects of T7L variants on E. coli metabolic profiles, with T7L-H48K showing minimal alterations in energy and amino acid pathways linked to osmotic stress compared to noticeable alterations in these pathways for both T7L-H37A and T7L-WT.
CONCLUSIONS
This study uncovered distinct metabolic fingerprints when comparing the overexpression of active and inactive mutants of T7L lytic enzymes in E. coli cells. These findings could contribute to the optimization and enhancement of suitable endolysins as potential alternatives to antibiotics.
Topics: Escherichia coli; Metabolome; Metabolomics; Viral Proteins; Bacteriophage T7; Mutation; DNA-Directed RNA Polymerases
PubMed: 38941046
DOI: 10.1007/s11306-024-02133-y -
BdRCN4, a Brachypodium distachyon TFL1 homologue, is involved in regulation of apical meristem fate.Plant Molecular Biology Jun 2024In higher plants, the shift from vegetative to reproductive development is governed by complex interplay of internal and external signals. TERMINALFLOWER1 (TFL1) plays a...
In higher plants, the shift from vegetative to reproductive development is governed by complex interplay of internal and external signals. TERMINALFLOWER1 (TFL1) plays a crucial role in the regulation of flowering time and inflorescence architecture in Arabidopsis thaliana. This study aimed to explore the function of BdRCN4, a homolog of TFL1 in Brachypodium distachyon, through functional analyses in mutant and transgenic plants. The results revealed that overexpression of BdRCN4 in B. distachyon leads to an extended vegetative phase and reduced production of spikelets. Similar results were found in A. thaliana, where constitutive expression of BdRCN4 promoted a delay in flowering time, followed by the development of hypervegetative shoots, with no flowers or siliques produced. Our results suggest that BdRCN4 acts as a flowering repressor analogous to TFL1, negatively regulating AP1, but no LFY expression. To further validate this hypothesis, a 35S::LFY-GR co-transformation approach on 35::BdRCN4 lines was performed. Remarkably, AP1 expression levels and flower formation were restored to normal in co-transformed plants when treated with dexamethasone. Although further molecular studies will be necessary, the evidence in B. distachyon support the idea that a balance between LFY and BdRCN4/TFL1 seems to be essential for activating AP1 expression and initiating floral organ identity gene expression. This study also demonstrates interesting conservation through the molecular pathways that regulate flowering meristem transition and identity across the evolution of monocot and dicot plants.
Topics: Brachypodium; Meristem; Gene Expression Regulation, Plant; Plant Proteins; Flowers; Plants, Genetically Modified; Arabidopsis; Arabidopsis Proteins
PubMed: 38940986
DOI: 10.1007/s11103-024-01467-4 -
Membrane protein Bcsdr2 mediates biofilm integrity, hyphal growth and virulence of Botrytis cinerea.Applied Microbiology and Biotechnology Jun 2024Grey mould caused by Botrytis cinerea is a devastating disease responsible for large losses to agricultural production, and B. cinerea is a necrotrophic model fungal...
Grey mould caused by Botrytis cinerea is a devastating disease responsible for large losses to agricultural production, and B. cinerea is a necrotrophic model fungal plant pathogen. Membrane proteins are important targets of fungicides and hotspots in the research and development of fungicide products. Wuyiencin affects the permeability and pathogenicity of B. cinerea, parallel reaction monitoring revealed the association of membrane protein Bcsdr2, and the bacteriostatic mechanism of wuyiencin was elucidated. In the present work, we generated and characterised ΔBcsdr2 deletion and complemented mutant B. cinerea strains. The ΔBcsdr2 deletion mutants exhibited biofilm loss and dissolution, and their functional activity was illustrated by reduced necrotic colonisation on strawberry and grape fruits. Targeted deletion of Bcsdr2 also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted gene complementation. The roles of Bcsdr2 in biofilms and pathogenicity were also supported by quantitative real-time RT-PCR results showing that phosphatidylserine decarboxylase synthesis gene Bcpsd and chitin synthase gene BcCHSV II were downregulated in the early stages of infection for the ΔBcsdr2 strain. The results suggest that Bcsdr2 plays important roles in regulating various cellular processes in B. cinerea. KEY POINTS: • The mechanism of wuyiencin inhibits B. cinerea is closely associated with membrane proteins. • Wuyiencin can downregulate the expression of the membrane protein Bcsdr2 in B. cinerea. • Bcsdr2 is involved in regulating B. cinerea virulence, growth and development.
Topics: Botrytis; Biofilms; Virulence; Hyphae; Plant Diseases; Fragaria; Fungal Proteins; Membrane Proteins; Vitis; Spores, Fungal; Gene Deletion
PubMed: 38940906
DOI: 10.1007/s00253-024-13238-8 -
Antimicrobial Agents and Chemotherapy Jun 2024Intrinsic resistance to macrolides in Gram-negative bacteria is primarily attributed to the low permeability of the outer membrane, though the underlying genetic and...
Intrinsic resistance to macrolides in Gram-negative bacteria is primarily attributed to the low permeability of the outer membrane, though the underlying genetic and molecular mechanisms remain to be fully elucidated. Here, we used transposon directed insertion-site sequencing (TraDIS) to identify chromosomal non-essential genes involved in intrinsic resistance to a macrolide antibiotic, tilmicosin. We constructed two highly saturated transposon mutant libraries of >290,000 and >390,000 unique Tn5 insertions in a clinical enterotoxigenic strain (ETEC5621) and in a laboratory strain (K-12 MG1655), respectively. TraDIS analysis identified genes required for growth of ETEC5621 and MG1655 under 1/8 MIC ( = 15 and 16, respectively) and 1/4 MIC ( = 38 and 32, respectively) of tilmicosin. For both strains, 23 genes related to lipopolysaccharide biosynthesis, outer membrane assembly, the Tol-Pal system, efflux pump, and peptidoglycan metabolism were enriched in the presence of the antibiotic. Individual deletion of genes ( = 10) in the wild-type strains led to a 64- to 2-fold reduction in MICs of tilmicosin, erythromycin, and azithromycin, validating the results of the TraDIS analysis. Notably, deletion of or , which impairs the outer membrane, led to the most significant decreases in MICs of all three macrolides in ETEC5621. Our findings contribute to a genome-wide understanding of intrinsic macrolide resistance in , shedding new light on the potential role of the peptidoglycan layer. They also provide an proof of concept that can be sensitized to macrolides by targeting proteins maintaining the outer membrane such as SurA and WaaG.
PubMed: 38940570
DOI: 10.1128/aac.00452-24 -
Development (Cambridge, England) Jul 2024During heart development, the embryonic ventricle becomes enveloped by the epicardium, which adheres to the outer apical surface of the heart. This is concomitant with...
During heart development, the embryonic ventricle becomes enveloped by the epicardium, which adheres to the outer apical surface of the heart. This is concomitant with onset of ventricular trabeculation, where a subset of cardiomyocytes lose apicobasal polarity and delaminate basally from the ventricular wall. Llgl1 regulates the formation of apical cell junctions and apicobasal polarity, and we investigated its role in ventricular wall maturation. We found that llgl1 mutant zebrafish embryos exhibit aberrant apical extrusion of ventricular cardiomyocytes. While investigating apical cardiomyocyte extrusion, we identified a basal-to-apical shift in laminin deposition from the internal to the external ventricular wall. We find that epicardial cells express several laminin subunits as they adhere to the ventricle, and that the epicardium is required for laminin deposition on the ventricular surface. In llgl1 mutants, timely establishment of the epicardial layer is disrupted due to delayed emergence of epicardial cells, resulting in delayed apical deposition of laminin on the ventricular surface. Together, our analyses reveal an unexpected role for Llgl1 in correct timing of epicardial development, supporting integrity of the ventricular myocardial wall.
Topics: Animals; Laminin; Zebrafish; Pericardium; Heart Ventricles; Zebrafish Proteins; Myocytes, Cardiac; Cell Polarity; Mutation
PubMed: 38940292
DOI: 10.1242/dev.202482 -
Journal of Extracellular Biology Aug 2023Non-coding RNAs (ncRNAs) are important regulators of gene expression. They are expressed not only in cells, but also in cell-derived extracellular vesicles (EVs). The...
Non-coding RNAs (ncRNAs) are important regulators of gene expression. They are expressed not only in cells, but also in cell-derived extracellular vesicles (EVs). The mechanisms controlling their loading and sorting remain poorly understood. Here, we investigated the impact of mutations on the non-coding RNA content of small melanoma EVs. After purification of small EVs from six different patient-derived melanoma cell lines, we characterized them by small RNA sequencing and lncRNA microarray analysis. We found that mutations are associated with a specific micro and long non-coding RNA content in small EVs. Then, we showed that long and small non-coding RNAs enriched in mutant small EVs share a common sequence motif, highly similar to the RNA-binding motif of Sam68, a protein interacting with hnRNP proteins. This protein thus may be an interesting partner of p53, involved in the expression and loading of the ncRNAs. To conclude, our data support the existence of cellular mechanisms associate with mutations which control the ncRNA content of small EVs in melanoma.
PubMed: 38939511
DOI: 10.1002/jex2.105 -
Journal of Experimental Botany Jun 2024Raffinose mitigates plant heat-, drought- and cold- stresses; however, whether raffinose contributes to plant waterlogging tolerance is unknown. The maize zmrafs-1...
Raffinose mitigates plant heat-, drought- and cold- stresses; however, whether raffinose contributes to plant waterlogging tolerance is unknown. The maize zmrafs-1 mutant seedlings lacking raffinose, generate fewer and shorter adventitious root (AR) and are more sensitive to waterlogging stress, while overexpression of ZmRAFS increases raffinose content, stimulates AR formation, and enhances the waterlogging tolerance of maize seedlings. Transcriptome analysis of NS (Null segregant) seedlings compared with that of zmrafs-1, particularly when waterlogged, revealed that the expression of genes related to galactose metabolism and the auxin biosynthetic pathway were upregulated by raffinose. Additionally, Indole-3-acetic acid (IAA) amounts significantly decreased or increased in zmrafs-1 or ZmRAFS-overexpressing seedlings, respectively. Inhibition of the hydrolysis of raffinose by DGJ (1-deoxygalactonojirimycin) decreased the waterlogging tolerance of maize seedlings, decreased the expression of genes encoding proteins related to auxin transport-related genes as well as the IAA level in the seedlings, suggesting that the hydrolysis of raffinose is necessary for maize waterlogging tolerance. These data demonstrate that raffinose catabolism stimulates adventitious root formation via auxin signaling pathway to enhance maize waterlogging tolerance.
PubMed: 38938017
DOI: 10.1093/jxb/erae284 -
Virulence Dec 2024causes globally prevalent infections that are highly related to chronic gastritis and even development of gastric carcinomas. With the increase of antibiotic...
causes globally prevalent infections that are highly related to chronic gastritis and even development of gastric carcinomas. With the increase of antibiotic resistance, scientists have begun to search for better vaccine design strategies to eradicate colonization. However, while current strategies prefer to formulate vaccines with a single antigen, their potential has not yet been fully realized. Outer membrane vesicles (OMVs) are a potential platform since they could deliver multiple antigens. In this study, we engineered three crucial antigen proteins (UreB, CagA, and VacA) onto the surface of OMVs derived from serovar Typhimurium (. Typhimurium) mutant strains using the hemoglobin protease (Hbp) autotransporter system. In various knockout strategies, we found that OMVs isolated from the Δ Δ Δ Δ mutants could cause distinct increases in immunoglobulin G (IgG) and A (IgA) levels and effectively trigger T helper 1- and 17-biased cellular immune responses, which perform a vital role in protecting against . Next, OMVs derived from Δ Δ Δ Δ mutants were used as a vector to deliver different combinations of antigens. The antibody and cytokine levels and challenge experiments in mice model indicated that co-delivering UreB and CagA could protect against and antigen-specific T cell responses. In summary, OMVs derived from the . Typhimurium Δ Δ Δ Δ mutant strain as the vector while importing UreB and CagA as antigenic proteins using the Hbp autotransporter system would greatly benefit controlling infection.
Topics: Animals; Helicobacter Infections; Bacterial Proteins; Helicobacter pylori; Mice; Salmonella typhimurium; Antigens, Bacterial; Bacterial Vaccines; Female; Antibodies, Bacterial; Immunoglobulin G; Genetic Engineering; Urease; Disease Models, Animal
PubMed: 38937901
DOI: 10.1080/21505594.2024.2367783