Sepsis-associated bacteria release ATP in a growth-dependent manner.

(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 (OD600) 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 (OD600) 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.

ATP release is dependent on ATP synthesis.

(A) Illustration depicting the location of ATP synthase and cytochrome bo3 oxidase in gramneg bacteria.

(B) Measurement of released ATP (M) and growth (OD600) over time (hours) from cytochrome bo3 oxidase (cyo) and ATP synthase (atp) mutants. The PS was added as a control. N = 2 independent bacteria cultures. Means and standard deviations are shown.

(C) Area under the curve (AUC) of released ATP over time (M*hours) of the previously assessed bacteria (cumulative ATP) is shown individually in the left panel. N = 2 independent bacteria cultures.

Means and individual values are shown. Means of grouped cyo and atp mutants are compared in the right panel. T-test. Means and individual values are shown.

(D) Cumulative ATP (M*hours) and cumulative growth (OD600*hours) of all assessed cyo and atp mutants and the PS were plotted against each other. Pearson’s correlation (r) and coefficient of determination (R2) of the applied linear model are depicted. 95% confidence level is shown by the black dashed lines.

Outer membrane integrity and bacterial death determine bacterial ATP release during growth.

(A) Illustration depicting the location of outer membrane porins in gramneg bacteria.

(B) Measurement of released ATP (M) and growth (OD600) over time (hours) from outer membrane porin mutants. The PS and the PS supplemented with either 1mM Ca2+ or 0.5mM EDTA were added as controls. N = 2 independent bacteria cultures. Means and standard deviations are shown. The red line marks the individual peak of ATP release and growth (OD600) at that time point.

(C) 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.

(D) ATP concentration (M) and growth (OD600) at the individual peak of ATP release of all assessed outer membrane porin mutants, the PS and the PS+Ca2+ (no peak for the EDTA control) were plotted against each other. Pearson’s correlation (r) and coefficient of determination (R2) of the applied linear model are depicted. 95% confidence level is shown by the black dashed lines.

(E) Gating strategy to identify added counting beads, live, injured and dead bacteria.

(F) Quantitative assessment of injured and dead bacteria, as identified by flow cytometry after 4h of culturing (ATP peak) of the PS, ΔompF and ΔompC. One-way ANOVA followed by Tukey post-hoc test, N = 4 independent bacteria cultures. Means and individual values are shown.

(G) ATP concentration (M) after 4h of culturing (ATP peak) of the PS, ΔompF and ΔompC. One-way ANOVA followed by Tukey post-hoc test, N = 2 independent bacteria cultures. Means and individual values are shown.

Bacterial ATP reduces neutrophil counts and impairs sepsis outcome in vivo.

(A) Experimental approach to determine the local role of bacterial ATP in vivo, i.a. injecting PS+pEMPTY or PS+pAPY.

(B) Measurement of released ATP (M) in bacteria culture supernatant immediately before bacteria were i.a. injected. T-test, N = 2 independent bacteria cultures. Means and individual values are shown.

(C) Measurement of ATP (M) in abdominal fluid from mice four hours after i.a. injection of bacteria. T-test, n = 5 animals per group of N = 2 independent experiments. Means and individual values are shown.

(D) Quantitative assessment of colony forming units in abdominal fluid and (E) blood from mice four hours after i.a. injection of bacteria. Wilcoxon rank sum test, n = 5 animals per group of N = 2 independent experiments. Means and individual values are shown. No growth for controls was detected.

(E) Kaplan-Meier curves of mice after i.a. injection of bacteria. Log-rank test, n = 10 animals per group.

(F) Heatmap showing surface marker expression (x-Axis), which was used to characterize the different immune cell populations (y-Axis).

(G) Concatenated (n = 5 animals for each treatment group, n = 3 animals for control group of N = 2 independent experiments) and down-sampled images of immune cell populations characterized in the abdominal cavity four hours after sham treatment or i.a. injection of bacteria.

(H) Abundance of neutrophils, small peritoneal macrophages (SPM) and CX3CR1pos monocytes in abdominal fluid from mice four hours after sham treatment or i.a. injection of bacteria. One-way ANOVA followed by Tukey post-hoc test, n = 5 animals for each treatment group, n = 3 animals for control group of N = 2 independent experiments. Means and individual values are shown.

OMV contain ATP and can be exploited as a model to assess the systemic relevance of bacterial ATP.

(A) Illustration depicting the location of assessed proteins that lead to a hypervesiculation phenotype if knocked-out in the gramneg bacterium E. coli.

(B) Relative amount of OMV compared to the PS isolated from growth cultures of the assessed hypervesiculation mutants after five hours (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 hours. 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. 20µl of E. coli growth culture and 20µl 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. Column 2-5: different concentrations of ATP incubated for one hour at 37°C (passive filling). Column 6-12: different voltages with fixed settings for Resistance (100Ω) and Capacitance (50µF). N = 2-9 independent experiments. Means and standard deviations are shown.

(G) Relative quantification of ATP in OMV over 24 hours at 37°C after electroporation (0h = 100%). N = 2 measurements of N = 3 independent experiments. Means and individual values are shown.

Bacterial ATP within OMV upregulates lysosome-related pathways and degranulation processes in neutrophils.

(A) Experimental approach to determine the systemic role of bacterial ATP in vivo, i.a. injecting ATP-loaded or empty OMV.

(B) Representative microscopic images of cells from the abdominal cavity one hour after i.a. injection of either ATP-loaded or empty OMV. OMV: DiI, Nucleus: DAPI, Neutrophils: Ly-6G-FITC.

(C) Cells from remote organs were isolated one hour after i.a. injection of either ATP-loaded or empty OMV. OMV were mainly taken up by neutrophils (except in the spleen, ratio ≈ 1). T-test with Benjamini-Hochberg correction, n = 5 animals per group of N = 2 independent experiments. Means and individual values are shown.

(D) Representative microscopic image of pulmonary neutrophils one hour after i.a. injection of either ATP-loaded or empty OMV. OMV co-localize with the endolysosomal compartment. OMV: DiI, Endolysosomal system: Deep Red LysoTracker, Neutrophils: Ly-6G-FITC.

(E) Pulmonary neutrophils were isolated one hour after i.a. injection of ATPγs-loaded or empty OMV, bead-sorted and RNA sequencing was done. Principal component analysis shows significantly different clustering between neutrophils that took up ATPγs-loaded (NA) or empty OMV (NE). PERMANOVA, n = 6 animals in the NE group, n = 5 animals in the NA group. Ellipses represent 95% confidence level.

(F) Volcano plot of RNA sequencing results shows an upregulation of genes mainly in the NA group. Genes classified in either lysosome (LYSO) or neutrophil degranulation pathways (NDG) or both, which were mentioned in the text, were highlighted.

(G) Heatmap of the lysosome pathway (LYSO) showing the gene expression per sample.

(H) Heatmap of the neutrophil degranulation pathway (NDG) showing the gene expression per sample.

Experimental approach to measure released bacterial ATP and growth over time.

Immune cell characterization eight hours after i.a. injection of bacteria.

(A) Measurement of released ATP (M) and growth (OD600) over time (hours) from PS+pEMPTY and PS+pAPY. n = 2 measurements of N = 3 independent bacteria cultures. Means and standard deviations are shown.

(B) Area under the curve (AUC) of released ATP over time (M*hours) of the previously assessed bacteria (cumulative ATP). T-test, n = 2 measurements of N = 3 independent bacteria cultures. Means and individual values are shown.

(C) Measurement of ATP (M) in abdominal fluid from mice eight hours after i.a. injection of bacteria. T-test, n = 5 animals per group of N = 2 independent experiments. Means and individual values are shown.

(D) Quantitative assessment of colony forming units in abdominal fluid and (E) blood from mice eight hours after i.a. injection of bacteria. Wilcoxon rank sum test, n = 5 animals per group of N = 2 independent experiments. Means and individual values are shown. No growth for controls was detected.

(E) Concatenated (n = 5 animals for each treatment group, n = 3 animals for control group of N = 2 independent experiments) and down-sampled images of immune cell populations characterized in the abdominal cavity eight hours after sham treatment or i.a. injection of bacteria.

(G) Abundance of neutrophils, small peritoneal macrophages (SPM) and CX3CR1pos monocytes in abdominal fluid from mice eight hours after sham treatment or i.a. injection of bacteria. One-way ANOVA followed by Tukey post-hoc test, n = 5 animals for each treatment group, n = 3 animals for control group of N = 2 independent experiments. Means and individual values are shown.

ATP measurement of the PS, ΔnlpI as well as ΔtolB and OMV collection and characterization.

(A) Measurement of released ATP (M) and growth (OD600) over time (hours) from PS, ΔnlpI and ΔtolB. OMV collection time points are marked in purple. n = 2 measurements of N = 3 independent bacteria cultures. Means and standard deviations are shown.

(B) OMV before and after density gradient ultracentrifugation for 16h at 150’000g.

(C) Statistical parameters of OMV before electroporation as well as ATP-loaded and empty OMV after electroporation.

(D) Relative quantification of ATP in OMV 16 hours at 4°C after electroporation (0h = 100%). n = 2 measurements of N = 3 independent experiments. Means and individual values are shown.

Uptake of OMV by neutrophils.

Representative images of OMV uptake by neutrophils in the abdominal cavity one hour after i.a. injection additionally assessed using flow cytometry (Image Stream).

Characterization of local immune response in the abdominal cavity.

(A) Gating strategy to identify large peritoneal macrophages (LPM), small peritoneal macrophages (SPM) and neutrophils in abdominal fluid.

(B) Abundance of OMVpos / (OMVpos+OMVneg) LPM, SPM and neutrophils one hour after i.a. injection of ATP-loaded or empty OMV. T-test with Benjamini-Hochberg correction, n = 5 animals per group of N = 2 independent experiments. Means and individual values are shown.

(C) Abundance of LPM, SPM and neutrophils one hour after sham treatment or i.a. injection of either ATP-loaded or empty OMV. One-way ANOVA, n = 5 animals for each treatment group, n = 3 animals for control group of N = 2 independent experiments. Means and individual values are shown.

Assessment of OMV uptake by immune cells in remote organs.

(A) Gating strategy to identify total OMVpos cells and specifically OMVpos neutrophils in blood and remote organs (lung, liver, kidney, and spleen)

(B) Abundance of OMVpos / (OMVpos+OMVneg) neutrophils one hour after i.a. injection of ATP-loaded or empty OMV. T-test, n = 5 animals per group of N = 2 independent experiments. Means and individual values are shown.

Assessment of the purity of bead-sorted pulmonary neutrophils.

Pulmonary neutrophils were isolated one hour after i.a. injection of ATPγs-loaded or empty OMV, bead-sorted and assessed for purity by flow cytometry. A representative image is shown.

List of significantly different pathways after enrichment analysis of RNA sequencing results.

Pulmonary neutrophils were isolated one hour after i.a. injection of ATPγs-loaded or empty OMV, bead-sorted and RNA sequencing was done. This resulted in these significantly different pathways between the groups after enrichment analysis. DESeq, n = 6 animals in the NE group, n = 5 animals in the NA group.