Journal: Nature Communications

Article Title: N-acetyl-glucosamine primes Pseudomonas aeruginosa for virulence through a type IV pili/cAMP-mediated morphology transition

doi: 10.1038/s41467-025-64071-0

Figure Lengend Snippet: a Survival of wild-type flies injected with a set of higBA mutants in PAO1. b Bacterial load corresponding to ( a ). c Survival of wild-type flies injected with a set of higBA overexpression strains. d Bacterial load corresponding to ( c ). e Survival of wild-type and key flies injected with ∆ higA . f Bacterial load corresponding to ( e ). g Survival of Group B AMPs-deficient flies injected with ∆ higA . Group B indicates flies lacking Diptericins, Drosocin/Buletin , and Attacins . h Survival of single AMP gene-deficient flies injected with ∆ higA . Bars represent the standard error of the mean ( a , c , e ) or the median ( b, d, f ). Fly and bacteria strains are denoted by distinct symbol shapes (filled circles represent wild-type flies, unfilled circles indicate key flies, filled squares denote Group B AMP-deficient flies, variant circle shapes ( e.g ., half-filled) represent individual AMP-deficient strains) and colors (wild type PAO1, Δ higA , Δ higB , Δ higA -Δ higB , PAO1 higA OE , PAO1 higB OE , PAO1 higBA OE were distinguished in turn by orange, dark purple, light blue, yellowish-green, dark red, light purple and indigo) respectively (hereinafter the same). Experiments were repeated three times ( a , c , e – h ; pooled data; n = 24 in f ), twice ( b ; pooled data; n = 18 at 18hpi, n = 12 at 48hpi), once ( d , n = 8). Statistical analysis was done by Logrank (Mantel-Cox test) in ( a , c , e , g , h ), by Kruskal-Wallis test with Dunn’s post-hoc test in ( b , d , f ). Source data are provided as a Source Data file.

Article Snippet: Wild type PAO1, Δ higA , Δ higB , Δ higBA mutants (each in three biological triplicates) cultured in BHI were harvested by centrifugation at exponential phase and put in dry ice for delivery to the Applied Protein Technology company (Shanghai, China).

Techniques: Injection, Over Expression, Bacteria, Variant Assay

Journal: Nature Communications

Article Title: N-acetyl-glucosamine primes Pseudomonas aeruginosa for virulence through a type IV pili/cAMP-mediated morphology transition

doi: 10.1038/s41467-025-64071-0

Figure Lengend Snippet: a Kinetics of Dpt steady-state transcript levels after P. aeruginosa injection. b Co-injection of PAO1 and ∆ higA in wild-type flies. c Bacterial load corresponding to ( b ). d, e Bacterial load in wild-type flies ( d ) and key flies ( e ) first injected with PAO1 gfp and then secondarily injected with PAO1 rfp 24 h later. Survival data are shown in Supplementary Fig. . f Virulence of retrieved bacteria. The fly genotypes enclosed by parentheses indicate the donors for retrieved bacteria. HK enclosed by parentheses refers to flies pre-challenged by heat-killed bacteria. g Survival of imd -overexpressing flies challenged with naive or retrieved bacteria. h Bacterial load corresponding to ( g ). Bars represent the median ( a, c, d, e, h ) or the standard error of the mean ( b, f, g ). The GFP and RFP labeled bacteria are denoted by light green and pink, respectively. PAO1 injected into imd OE flies were denoted by stars and distinguished by orange (PAO1 cultured) and ultramarine (PAO1 retrieved). Experiments were repeated three times ( a–h ; pooled data; n = 8 in a; n = 24 in c , d , e , h ). Statistical analysis was done by Logrank (Mantel-Cox test) in ( b, f–g ), by Kruskal-Wallis test with Dunn’s post-hoc test in ( a, c –e, h ). Source data are provided as a Source Data file.

Article Snippet: Wild type PAO1, Δ higA , Δ higB , Δ higBA mutants (each in three biological triplicates) cultured in BHI were harvested by centrifugation at exponential phase and put in dry ice for delivery to the Applied Protein Technology company (Shanghai, China).

Techniques: Injection, Bacteria, Labeling, Cell Culture

Journal: Nature Communications

Article Title: N-acetyl-glucosamine primes Pseudomonas aeruginosa for virulence through a type IV pili/cAMP-mediated morphology transition

doi: 10.1038/s41467-025-64071-0

Figure Lengend Snippet: a –c Characteristic morphology of P. aeruginosa PAO1 and Δ higA in vitro, in wild-type or key flies. Because of the limited number of bacterial cells in most visual fields, all figures in vivo are composite assemblies from different visual fields. Morphology of PAO1 was visualized by transmission electron microscopy in vitro and in vivo ( a ). Morphology of PAO1 and Δ higA were visualized by O5 antibody staining under fluorescence microscopy in vitro, in tissues of wild-type or key flies in ( b ) and at different time points in ( c ). The lengths of bacteria and the length/width ratios of bacteria were quantified respectively in ( a’, b’, c’ ) and ( a” ). d Survival of flies infected by lysozyme treated P. aeruginosa . EDTA, ethylene diamine tetraacetic acid; Lz, lysozyme. e Survival of flies injected first with chitinase and then with P. aeruginosa 30 min later. f –h Survival of flies co-injected with P. aeruginosa in a N-acetyl-glucosamine (NAG)-containing solution ( f ), NAG- and N-acetyl-muramic acid (NAM)-containing solution ( g ) and NAM-containing solution ( h ). Bars represent the mean ( a, a’, b’, c’ ) or the standard error of the mean ( d – h ). The treatments of bacteria are denoted by distinct colors (Lz treatment: bright blue; chitinase treatment: yellowish-blue; NAG supplementation: light blue; NAG and NAM supplemented: pink; NAM supplemented: sky blue; hereinafter the same color code is used). Experiments were repeated three times ( b – h ; pooled data; For panel b’ , the following data points were analyzed: (i) PAO1 in vitro ( n = 64), WT flies ( n = 36), and key flies ( n = 11); (ii) Δ higA in vitro ( n = 25), WT flies ( n = 65), and key flies ( n = 27). For panel c’ , the following data points were analyzed: 13hpi ( n = 14), 16hpi ( n = 86)), once ( a-a ”; n = 5). Statistical analysis was done by one tailed Student’s t-test in ( a’-a”, c’ ), by Kruskal-Wallis test with Dunn’s post-hoc test in ( b’ ), by Logrank (Mantel-Cox test) in ( d – h ). Source data are provided as a Source Data file.

Article Snippet: Wild type PAO1, Δ higA , Δ higB , Δ higBA mutants (each in three biological triplicates) cultured in BHI were harvested by centrifugation at exponential phase and put in dry ice for delivery to the Applied Protein Technology company (Shanghai, China).

Techniques: In Vitro, In Vivo, Transmission Assay, Electron Microscopy, Staining, Fluorescence, Microscopy, Bacteria, Infection, Injection, One-tailed Test

Journal: Nature Communications

Article Title: N-acetyl-glucosamine primes Pseudomonas aeruginosa for virulence through a type IV pili/cAMP-mediated morphology transition

doi: 10.1038/s41467-025-64071-0

Figure Lengend Snippet: a Heatmap of differentially expressed proteins in mutants affecting higBA based on in vitro proteomic analysis. b Survival of flies challenged by ∆ pilJ . c, d Survival of flies challenged by ∆ higA bacteria harboring a plasmid overexpressing pilJ ( c ) or cyaB ( d ). e Survival of flies challenged by different mutants related to the T4P, including ∆ pilJ , ∆ pilA , ∆ pilC , ∆ vfr . f Bacterial load corresponding to ( e ). g Morphology of ∆ vfr in the hemolymph wild-type and key flies visualized by O5 antibody staining under fluorescence microscope. Because of the limited number of bacterial cells in most visual fields, all figures are composites from different visual fields. g’ Quantitative analysis of cell length in ( g ). Bars represent the standard error of the mean ( b – e ), the median ( f ), or the mean ( g’ ). Bacteria strains are denoted by distinct colors (Δ pilJ and Δ higA - pilJ OE : brick red; Δ higA - cyaB OE : sky blue; Δ vfr : blue-gray; hereinafter the same color code is used). Experiments were repeated three times ( b-g’ ; pooled data; f ( n = 24); g’ : in WT flies ( n = 51), in key flies ( n = 50)); once with three replicates ( a ). Statistical analysis was done by Logrank (Mantel-Cox test) in ( b – e ), by two tailed Student’s t-test in ( f , g’ ). n.s.: not significant. Source data are provided as a Source Data file.

Article Snippet: Wild type PAO1, Δ higA , Δ higB , Δ higBA mutants (each in three biological triplicates) cultured in BHI were harvested by centrifugation at exponential phase and put in dry ice for delivery to the Applied Protein Technology company (Shanghai, China).

Techniques: In Vitro, Bacteria, Plasmid Preparation, Staining, Fluorescence, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: N-acetyl-glucosamine primes Pseudomonas aeruginosa for virulence through a type IV pili/cAMP-mediated morphology transition

doi: 10.1038/s41467-025-64071-0

Figure Lengend Snippet: a Survival of flies injected with ∆ pilJ in NAG-containing solution. b Characteristic morphology of ∆ pilJ bacteria with NAG co-injection in the tissues of wild-type and key flies visualized by O5 antibody staining under fluorescence microscope. Because of the limited number of bacterial cells in most visual fields, all figures are composites from different visual fields. b’ Quantitative analysis of cell length in ( b ). c Ligand binding assay was determined by thermal shift assay with NAG (blue lines) using protein without ligand as control (red lines). d Survival of flies challenged by ∆ pilJ harboring a plasmid overexpressing full-length pilJ , pilJ ΔLBD , or LBD alone. e Survival of flies injected with ∆ pilJ harboring a plasmid overexpressing pilJ ΔLBD in NAG-containing solution. f Kinetics of transcript levels of the readout gene pilA for cAMP-Vfr signaling in wild-type P. aeruginosa and Δ pilJ upon NAG in vivo. g Survival of flies challenged with ∆ pilJ in NAM-containing solution. h Ligand binding assay was determined by thermal shift assay with NAM (blue lines) using protein without ligand as control (red lines). i Transcript levels of the readout gene pilA for cAMP-Vfr signaling in wild-type P. aeruginosa and Δ pilJ upon NAM 10 hours post infection. j Survival of flies injected with wild-type P. aeruginosa and ∆ pilJ in NAM or NAM-NAG-containing solution. k Transcript levels of the readout gene pilA for cAMP-Vfr signaling in wild-type P. aeruginosa and Δ pilJ upon NAM 48 h post infection. Bars represent the standard error of the mean ( a, d-e, j ), the mean ( c’ ), or the median ( f, i, k ). Experiments were repeated three times ( a-b’, d-g, i –k ; pooled data; b’ : WT flies ( n = 64), key flies ( n = 67), WT flies with NAG ( n = 50), key flies with NAG ( n = 20); k : PAO1 groups ( n = 15), Δ pilJ groups ( n = 8)), twice in ( f , i ; pooled data; n = 7-9), while one representative result of three independent experiments with consistent findings was displayed in ( c , h ). Statistical analysis was done by Logrank (Mantel-Cox test) in ( a , d , e , g , j ), by Kruskal-Wallis test with Dunn’s post-hoc test in ( b’ , f , i , k ). n.s.: not significant. Source data are provided as a Source Data file.

Article Snippet: Wild type PAO1, Δ higA , Δ higB , Δ higBA mutants (each in three biological triplicates) cultured in BHI were harvested by centrifugation at exponential phase and put in dry ice for delivery to the Applied Protein Technology company (Shanghai, China).

Techniques: Injection, Bacteria, Staining, Fluorescence, Microscopy, Ligand Binding Assay, Thermal Shift Assay, Control, Plasmid Preparation, In Vivo, Infection

Journal: Nature Communications

Article Title: N-acetyl-glucosamine primes Pseudomonas aeruginosa for virulence through a type IV pili/cAMP-mediated morphology transition

doi: 10.1038/s41467-025-64071-0

Figure Lengend Snippet: a Survival of flies challenged by ∆ higA bacteria overexpressing fimV . b Characteristic morphology of ∆ higA bacteria overexpressing fimV in vitro and in vivo in wild-type or key tissues visualized by O5 antibody staining under fluorescence microscope. b’ Quantitative analysis of cell length. c Characteristic morphology of bacteria overexpressing FimV fused EGFP and the location of FimV-EGFP in P. aeruginosa in vitro and in tissues of wild-type flies. d Scheme of different domains of FimV protein. e Survival of flies challenged with ∆ higA harboring plasmids overexpressing full-length of fimV , fimV ΔlysM , fimV ΔCC , or fimV ΔCD contructs. f Characteristic morphology of bacteria overexpressing FimV ΔCD fused EGFP and the location of FimV ΔCD -EGFP in P. aeruginosa in vitro and in tissues of wild-type flies. f’ Quantitative analysis of cell length in ( c , f ). g FimV-EGFP localization observed by fluorescence microscope in vivo in the hemolymph of wild-type flies. g’-g” Quantitative analysis of cell length ( g’ ) and width in ( g” ). h Survival curves of flies injected with ∆ higA bacteria overexpressing cell division genes. i Survival curves of flies challenged with PAO1 bacteria overexpressing cell wall elongation genes. j Characteristic morphology of PAO1 bacteria overexpressing cell elongation genes in the hemolymph of wild-type or key flies. j’ Quantitative analysis of cell length. Because of the limited number of bacterial cells in most visual fields, all figures in vivo are composites from different visual fields ( b, c, f, g, j ). Bars represent the standard error of the mean ( a, e, h, i ) or the mean ( b’, f’, g’, g”, j’ ). Experiments were repeated three times ( a–c, e-j’ ; pooled data; b’ : in vitro ( n = 145), WT flies ( n = 37), key flies ( n = 37); f’ : FimV-EGFP in vitro ( n = 43), FimV-EGFP in WT flies ( n = 13), FimV ΔCD -EGFP in vitro ( n = 119), FimV ΔCD -EGFP in WT flies ( n = 38); g’-g” : no treatment ( n = 10), NAG treatment ( n = 10), NAM treatment ( n = 48); j’ : PAO1- zipA OE in vitro ( n = 79), WT flies ( n = 35), key flies ( n = 29), PAO1- ftsW OE in vitro ( n = 26), WT flies ( n = 36), key flies ( n = 28)). Statistical analysis was done by Logrank (Mantel-Cox test) in ( a, e, h –j ), by Kruskal-Wallis test with Dunn’s post-hoc test in ( b’, f’,g’-g”, i’ ). Source data are provided as a Source Data file.

Article Snippet: Wild type PAO1, Δ higA , Δ higB , Δ higBA mutants (each in three biological triplicates) cultured in BHI were harvested by centrifugation at exponential phase and put in dry ice for delivery to the Applied Protein Technology company (Shanghai, China).

Techniques: Bacteria, In Vitro, In Vivo, Staining, Fluorescence, Microscopy, Injection