Journal: Veterinary Research

Article Title: Outer membrane vesicles secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing endoplasmic reticulum stress-mediated autophagy flux blockade

doi: 10.1186/s13567-025-01679-6

Figure Lengend Snippet: APEC leads to systemic infection by secreting OMVs to evade macrophage immune clearance . A Flowchart of chick infection. B Hematoxylin and eosin (HE) staining of the trachea, lung, liver, and spleen tissues from chicks infected with WT or WTΔ ypjA (1 × 10 9 CFU/chick) on day 6 post-infection. Trachea: degeneration and hyperplasia of epithelial mucosal cells (black arrow), diffuse neutrophilic infiltration (red arrow), tracheal wall with mucus and inflammatory exudate (blue arrow); lung: widened pulmonary septa (black arrow), inflammatory cell infiltration in the interstitium (red arrow); liver: hepatocytes show lytic degeneration (black arrow) with inflammatory cell infiltration (red arrow); spleen: hyperplasia of lymphoid follicles (black arrow), blurred demarcation between white and red pulp (red arrow). Scale bar, 50 µm or 200 µm. C , D Bacterial loads in trachea, lungs, liver, and spleen at day 3 ( C ) and day 6 ( D ) post-infection. C (95% CI): trachea: [0.31–0.42]; lungs: [0.43–0.54]; liver: [0.53–0.64]; spleen: [0.04–0.16]. D (95% CI): trachea: [0.52–0.65]; lungs: [0.36–0.49]; liver: [0.06–0.19]; spleen: [0.19–0.32]. E , F Intracellular survival of WT and WTΔ ypjA after infection of HD11 cells. Square culture plates represent 1/100 bacterial load ( n = 3). G , H Analysis of OMV morphology and concentration. Scale bar, 200 nm. I Western blot analysis of OmpA and OmpF proteins in APEC lysates, supernatant, and OMVs. J Confocal imaging of DiO-labeled OMVs entering HD11 cells; no signal in dye control. Representative of three experiments. Scale bar, 7.5 μm. n represents three biological replicates; data points indicate independent cultures. Bar charts show mean ± standard error of the mean (SEM) following two-way ANOVA with Sidak correction (* p < 0.05, ** p < 0.01, *** p < 0.001). Effect sizes with 95% CIs are reported in panels ( C ) and ( D ).

Article Snippet: Rabbit anti-OmpF polyclonal antibody , Biorbyt, Cambridge, UK , 1:500.

Techniques: Infection, Staining, Concentration Assay, Western Blot, Imaging, Labeling, Control

Journal: bioRxiv

Article Title: The spike tip protein of bacteriophage T4

doi: 10.1101/2025.08.28.672839

Figure Lengend Snippet: Flowchart of the fractionation procedure. The same nomenclature (colors and abbreviations) is used to label the cellular fractions in panels C and D. B , The two top rows in the left panel show titration of P2 vir1 Vam46 on either C-520 (permissive, supD ) or C-2 (non-permissive) E. coli lawns. The two bottom rows in the left panel show titration of P2 vir1 Vam46 that remained in solution after a 15 min incubation with cells. These cells were subsequently used in the fractionation procedure. The right panel shows quantitative representation (mean ± SD) of the titration presented in the left panel. The experiment was repeated six times with similar outcomes. C , Western blot analysis of cell fractions from cultures which were either uninfected (left section of the blot) or infected with P2 Vir1 V am46 (right section). Each column is a separate fraction of the fractionation procedure shown in panel A . The rows correspond to different antibodies used against cellular proteins with known localizations: GroEL is a soluble cytoplasmic protein, MalE (MBP, maltose binding protein) is a soluble periplasmic protein, OmpF is the outer membrane porin F. GpV co-localizes with MalE. The blot is representative of four biological replicates. D , A Coomassie stained polyacrylamide SDS gel showing a purified P2 Vir1 phage sample (labeled P2) and fractionated lysates of uninfected C-2 cells (labeled N) or infected with P2 Vir1 Vam (labeled I). The red arrow points to the P2 sheath protein; its identity was confirmed by LC/MS/MS analysis. The P2 capsid protein is partially masked by a cellular membrane component with a similar electrophoretic mobility. The inset in a black box (lower right) shows qPCR analysis of P2 genomic DNA (mean ± SD) found in different cellular fractions. The fluorescent signal was converted to the number of plaque forming units (pfus) using a calibration curve as described in the methods section. The experiment was repeated three times with similar outcomes. The significance was determined by Student’s two-tailed t -test with one, two, and three stars (*, **, ***) corresponding to p-values of less than than 0.05, 0.001, and 0.0001, respectively.

Article Snippet: The primary antibodies used were: MalE/MBP-probe antibody (Santa Cruz Biotechnology, sc-13564), GroEL polyclonal antibody (Enzo Life Sciences, ADI-SPS-875-D), OmpF antibody (orb308741, Biorbyt) and a custom made anti-P2 gpV polyclonal antibody generated by GenScript.

Techniques: Fractionation, Titration, Incubation, Western Blot, Infection, Binding Assay, Membrane, Staining, SDS-Gel, Purification, Labeling, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: eLife

Article Title: Released bacterial ATP shapes local and systemic inflammation during abdominal sepsis

doi: 10.7554/eLife.96678

Figure Lengend Snippet: ( A ) Illustration depicting the location of assessed proteins that lead to a hypervesiculation phenotype if knocked out in the gram neg bacterium E. coli . ( B ) Relative amount of OMV compared to the parental strain (PS) isolated from growth cultures of the assessed hypervesiculation mutants after 5 hr (exponential growth phase) and O/N (stationary phase). n=2 measurements of N=3 independent bacteria cultures. Means and individual values are shown. ( C ) Absolute quantification of ATP in OMV isolated from growth cultures of the PS, ΔnlpI and ΔtolB at their individual peak of ATP release and after 24 hr. n=2 measurements of N=3 independent bacteria cultures. Means and individual values are shown. ( D ) Amount of protein (BCA assay) detected in different fractions after density gradient ultracentrifugation. n=2 measurements of the different fractions. 20 µl of E. coli growth culture and 20 µl of each fraction were then characterized by Coomassie blue staining and specific detection of outer membrane ompF and cytoplasmic ftsZ. ( E ) Characterization of OMV by nanoparticle tracking analysis (n=5 measurements per sample) and electron microscopy (representative image) before and after electroporation. ( F ) Absolute quantification of ATP in OMV, which were loaded using different strategies. Columns 2–5: different concentrations of ATP incubated for 1 hr at 37°C (passive filling). Columns 6–12: different voltages with fixed settings for resistance (100 Ω) and capacitance (50 µF). N=2–9 independent experiments. Means and standard deviations are shown. ( G ) Relative quantification of ATP in OMV over 24 hr at 37°C after electroporation (0 hr=100%). n=2 measurements of N=3 independent experiments. Means and individual values are shown.

Article Snippet: Antibody , Rabbit anti- E. coli ompF, polyclonal , Biorbyt , Cat# orb308741; RRID: NA , (1:500).

Techniques: Isolation, Bacteria, Quantitative Proteomics, BIA-KA, Staining, Membrane, Electron Microscopy, Electroporation, Incubation

Journal: eLife

Article Title: Released bacterial ATP shapes local and systemic inflammation during abdominal sepsis

doi: 10.7554/eLife.96678

Figure Lengend Snippet: ( A ) Experimental approach to isolate and cultivate sepsis-associated bacteria from abdominal fluid of patients with abdominal sepsis. ( B ) Bacterial species identified by whole 16S-rRNA Sanger sequencing from abdominal fluid of patients with abdominal sepsis. Three colonies out of 25 could not be identified. ( C ) Measurement of released ATP (M) and growth (OD 600 ) over time (hours) from the four sepsis-associated bacteria E. coli , K. pneumoniae , E. faecalis, and S. aureus isolated from patients. N=2 independent bacteria cultures. Means and standard deviations are shown. ( D ) Area under the curve (AUC) of released ATP over time (M*hours) of the previously assessed bacteria (cumulative ATP). One-way ANOVA, N=2 independent bacteria cultures. Means and individual values are shown. ( E ) Experimental approach to isolate and cultivate sepsis-associated bacteria from abdominal fluid of mice with abdominal sepsis. ( F ) Bacterial species identified by whole 16S-rRNA Sanger sequencing from abdominal fluid of mice with abdominal sepsis. Seven colonies out of 25 could not be identified. ( G ) Measurement of released ATP (M) and growth (OD 600 ) over time (hours) from the three sepsis-associated bacteria E. coli , E. faecalis, and S. aureus isolated from mice. N=2 independent bacteria cultures. Means and standard deviations are shown. ( H ) AUC of released ATP over time (M*hours) of the previously assessed bacteria (cumulative ATP). One-way ANOVA, N=2 independent bacteria cultures. Means and individual values are shown.

Article Snippet: Antibody , Rabbit anti- E. coli ompF, polyclonal , Biorbyt , Cat# orb308741; RRID: NA , (1:500).

Techniques: Bacteria, Sequencing, Isolation

Journal: eLife

Article Title: Released bacterial ATP shapes local and systemic inflammation during abdominal sepsis

doi: 10.7554/eLife.96678

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit anti- E. coli ompF, polyclonal , Biorbyt , Cat# orb308741; RRID: NA , (1:500).

Techniques: Purification, Recombinant, Plasmid Preparation, Sequencing, Gel Extraction, Software