Released bacterial ATP shapes local and systemic inflammation during abdominal sepsis
- Department of Visceral Surgery and Medicine, Inselspital, Bern University Hospital, University Hospital of Bern, Switzerland
- Department for BioMedical Research, Visceral Surgery and Medicine, University Hospital of Bern, Switzerland
Figures
Figure 1 with 1 supplement
Sepsis-associated bacteria release adenosine triphosphate (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 …
Figure 1—figure supplement 1
Experimental approach to measure released bacterial adenosine triphosphate (ATP) and growth over time.
Figure 2 with 1 supplement
Adenosine triphosphate (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 …
Figure 2—figure supplement 1
Peak ATP (M) and peak 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.
Figure 3
Outer membrane integrity and bacterial death determine bacterial adenosine triphosphate (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 …
Figure 4 with 1 supplement
Bacterial adenosine triphosphate (ATP) reduces neutrophil counts and impairs sepsis outcome in vivo.
(A) Experimental approach to determine the local role of bacterial ATP in vivo, intraabdominal (i.a.) injecting parental strain (PS)+pEMPTY or PS+pAPY. (B) Measurement of released ATP (M) in …
Figure 4—figure supplement 1
Immune cell characterization 8 hr after intraabdominal (i.a.) injection of bacteria.
(A) Measurement of released adenosine triphosphate (ATP) (M) and growth (OD600) over time (hours) from parental strain (PS)+pEMPTY and PS+pAPY. n=2 measurements of N=3 independent bacteria cultures. …
Figure 5 with 1 supplement
Outer membrane vesicles (OMV) contain adenosine triphosphate (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 …
Figure 5—figure supplement 1
Adenosine triphosphate (ATP) measurement of the parental strain (PS), ΔnlpI as well as ΔtolB and outer membrane vesicle (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 …
Figure 6 with 5 supplements
Bacterial adenosine triphosphate (ATP) within outer membrane vesicles (OMV) upregulates lysosome-related pathways and degranulation processes in neutrophils.
(A) Experimental approach to determine the systemic role of bacterial ATP in vivo, intraabdominal (i.a.) injecting ATP-loaded or empty OMV. (B) Representative microscopic images of cells from the …
Figure 6—figure supplement 1
Uptake of outer membrane vesicles (OMV) by neutrophils.
Representative images of OMV uptake by neutrophils in the abdominal cavity 1 hr after intraabdominal (i.a.) injection additionally assessed using flow cytometry (ImageStream).
Figure 6—figure supplement 2
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 …
Figure 6—figure supplement 3
Assessment of outer membrane vesicle (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) Fraction of OMVpos/(OMVpos+OMVneg) …
Figure 6—figure supplement 4
Assessment of the purity of bead-sorted pulmonary neutrophils.
Pulmonary neutrophils were isolated 1 hr after i.a. injection of ATPγs-loaded or empty outer membrane vesicle (OMV), bead-sorted and assessed for purity by flow cytometry. A representative image is …
Figure 6—figure supplement 5
List of significantly different pathways after enrichment analysis of RNA sequencing results.
Pulmonary neutrophils were isolated 1 hr after intraabdominal (i.a.) injection of ATPγs-loaded or empty outer membrane vesicle (OMV), bead-sorted, and RNA sequencing was done. This resulted in these …
Author response image 1
Author response image 2
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus, female) | Wild-type mice | Inotiv, the Netherlands | C57Bl/6JRccHsd | |
| Strain, strain background (Enterococcus faecalis) | Iso1 | This paper | NA | See Materials and methods: Human data |
| Strain, strain background (Escherichia coli) | Iso2 | This paper | NA | See Materials and methods: Human data |
| Strain, strain background (Klebsiella pneumoniae) | Iso3 | This paper | NA | See Materials and methods: Human data |
| Strain, strain background (Staphylococcus aureus) | Iso4 | This paper | NA | See Materials and methods: Human data |
| Strain, strain background (E. faecalis) | Iso5 | This paper | NA | See Materials and methods: CLP sepsis model |
| Strain, strain background (E. coli) | Iso6 | This paper | NA | See Materials and methods: CLP sepsis model |
| Strain, strain background (S. aureus) | Iso7 | This paper | NA | See Materials and methods: CLP sepsis model |
| Strain, strain background (E. coli) | Parental strain (PS) | Keio collection; Baba et al., 2006 | BW25113 | |
| Strain, strain background (E. coli) | ΔcyoA | Keio collection; Baba et al., 2006 | JW0422-1 | See Figure 2 |
| Strain, strain background (E. coli) | ΔcyoB | Keio collection; Baba et al., 2006 | JW0421-1 | See Figure 2 |
| Strain, strain background (E. coli) | ΔcyoC | Keio collection; Baba et al., 2006 | JW0420-1 | See Figure 2 |
| Strain, strain background (E. coli) | ΔcyoD | Keio collection; Baba et al., 2006 | JW0419-1 | See Figure 2 |
| Strain, strain background (E. coli) | ΔatpA | Keio collection; Baba et al., 2006 | JW3712-1 | See Figure 2 |
| Strain, strain background (E. coli) | ΔatpB | Keio collection; Baba et al., 2006 | JW3716-1 | See Figure 2 |
| Strain, strain background (E. coli) | ΔatpC | Keio collection; Baba et al., 2006 | JW3709-2 | See Figure 2 |
| Strain, strain background (E. coli) | ΔatpD | Keio collection; Baba et al., 2006 | JW3710-1 | See Figure 2 |
| Strain, strain background (E. coli) | ΔatpE | Keio collection; Baba et al., 2006 | JW3715-1 | See Figure 2 |
| Strain, strain background (E. coli) | ΔatpF | Keio collection; Baba et al., 2006 | JW3714-2 | See Figure 2 |
| Strain, strain background (E. coli) | ΔatpH | Keio collection; Baba et al., 2006 | JW3713-1 | See Figure 2 |
| Strain, strain background (E. coli) | ΔompF | Keio collection; Baba et al., 2006 | JW0912-1 | See Figure 3 |
| Strain, strain background (E. coli) | ΔompC | Keio collection; Baba et al., 2006 | JW2203-1 | See Figure 3 |
| Strain, strain background (E. coli) | ΔlamB | Keio collection; Baba et al., 2006 | JW3996-1 | See Figure 3 |
| Strain, strain background (E. coli) | ΔphoE | Keio collection; Baba et al., 2006 | JW0231-1 | See Figure 3 |
| Strain, strain background (E. coli) | ΔmlaA | Keio collection; Baba et al., 2006 | JW2343-1 | See Figure 5 |
| Strain, strain background (E. coli) | ΔmlaE | Keio collection; Baba et al., 2006 | JW3161-1 | See Figure 5 |
| Strain, strain background (E. coli) | ΔnlpI | Keio collection; Baba et al., 2006 | JW3132-1 | See Figure 5 |
| Strain, strain background (E. coli) | ΔtolB | Keio collection; Baba et al., 2006 | JW5100-1 | See Figure 5 |
| Strain, strain background (E. coli) | ΔdegP | Keio collection; Baba et al., 2006 | JW0157-1 | See Figure 5 |
| Strain, strain background (E. coli) | ΔrfaD | Keio collection; Baba et al., 2006 | JW3594-1 | See Figure 5 |
| Strain, strain background (E. coli) | ΔrodZ | Keio collection; Baba et al., 2006 | JW2500-1 | See Figure 5 |
| Antibody | Purified anti-Ms CD16/32, monoclonal | BioLegend | Cat# 101302; clone 93; Lot# B298973; RRID: AB_312801 | (1:200) |
| Antibody | Rat anti-Ms Ly-6G (FITC), monoclonal | BD Biosciences | Cat# 551460; clone 1A8; Lot# 9068981; RRID: AB_394207 | (1:100) |
| Antibody | Rat anti-Ms Ly-6C (PerCP-Cyanine5.5), monoclonal | Thermo Fisher Scientific | Cat# 45-5932-82; clone HK1.4; Lot# 2309273; RRID: AB_2723343 | (1:100) |
| Antibody | Rat anti-Ms/Hs CD11b (APC), monoclonal | BioLegend | Cat# 101212; clone M1/70; Lot# B312600; RRID: AB_312795 | (1:800) |
| Antibody | Rat anti-Ms CD206 (AF700), monoclonal | BioLegend | Cat# 141733; clone C068C2; Lot# B278058; RRID: AB_2629636 | (1:300) |
| Antibody | Armenian hamster anti-Ms CD11c (APC-eFluor780), monoclonal | Thermo Fisher Scientific | Cat# 47-0114-80; clone N418; Lot# 2133269; RRID: AB_1548652 | (1:300) |
| Antibody | Rat anti-Ms CD45 (efluor450), monoclonal | Thermo Fisher Scientific | Cat# 48-0451-82; clone 30-F11; Lot# 2005853 RRID: AB_1518806 | (1:600) |
| Antibody | Rat anti-Ms CD19 (Super Bright 600), monoclonal | Thermo Fisher Scientific | Cat# 63-0193-82; clone eBio1D3; Lot# 2366423; RRID: AB_2637308 | (1:150) |
| Antibody | Rat anti-Ms CD3 (BV 605), monoclonal | BioLegend | Cat# 100237; clone 17A2; Lot# B389899; RRID: AB_2562039 | (1:100) |
| Antibody | Mouse anti-Ms NK1.1 (BV605), monoclonal | BioLegend | Cat# 108739; clone PK-136; Lot# B389899; RRID: AB_2562273 | (1:150) |
| Antibody | Rat anti-Ms CCR2 (BV650), monoclonal | BioLegend | Cat# 150613; clone SA203G11; Lot# B294599; RRID: AB_2721553 | (1:100) |
| Antibody | Rat anti-Ms I-A/I-E (BV711), monoclonal | BioLegend | Cat# 107643; clone M5/114.15.2; Lot# B299330; RRID: AB_2565976 | (1:600) |
| Antibody | Mouse anti-Ms CX3CR1 (BV785), monoclonal | BioLegend | Cat# 149029; clone SA011F11; Lot# B304744; RRID: AB_2565938 | (1:300) |
| Antibody | Rat anti-Ms Siglec F (PE), monoclonal | Thermo Fisher Scientific | Cat# 12-1702-80; clone 1RNM44N; Lot# 2252684; RRID: AB_2637129 | (1:300) |
| Antibody | Armenian hamster anti-Ms FcεR1α (PE/Dazzle 594), monoclonal | BioLegend | Cat# 134331; clone MAR-1; Lot# B280348; RRID: AB_2687240 | (1:300) |
| Antibody | Rat anti-Ms CD115 (PE-Cy7), monoclonal | BioLegend | Cat# 135523; clone AFS98; Lot# B268547; RRID: AB_2566459 | (1:600) |
| Antibody | Rat anti-Ms F4/80 (BUV395), monoclonal | BD Biosciences | Cat# 565614; clone T45-2342; Lot# 1104580; RRID: AB_2739304 | (1:150) |
| Antibody | Rat anti-Ms CD45 (APC-Cy7), monoclonal | BioLegend | Cat# 103115; clone 30-F11; Lot# NA; RRID: AB_312980 | (1:150) |
| Antibody | Rat anti-Ms Ly-6C (PE-Cy7), monoclonal | BioLegend | Cat# 128017; clone HK1.4; Lot# B331355; RRID: AB_1732093 | (1:600) |
| Antibody | Rat anti-Ms Ly-6G (Biotin), monoclonal | BioLegend | Cat# 127604; clone 1A8; Lot# B314606; RRID: AB_1186108 | (1:600) |
| Antibody | Rabbit anti-E. coli ftsZ, polyclonal | Agrisera | Cat# AS10715; RRID: AB_10754647 | (1:200) |
| Antibody | Rabbit anti-E. coli ompF, polyclonal | Biorbyt | Cat# orb308741; RRID: NA | (1:500) |
| Antibody | Goat anti-rabbit, polyclonal | LI-COR Biosciences | Cat# 925-68021; RRID: AB_2713919 | (1:10,000) |
| Recombinant DNA reagent | pBAD28==pEMPTY (plasmid) | Proietti et al., 2019; Santapaola et al., 2006; Scribano et al., 2014 | Ampicillin resistance | |
| Recombinant DNA reagent | pHND10==pAPY (plasmid) | Proietti et al., 2019; Santapaola et al., 2006; Scribano et al., 2014 | Ampicillin resistance | |
| Sequence-based reagent | fD1 | Weisburg et al., 1991 | PCR primers | 5’-AGA-GTT-TGA-TCC-TGG-CTC-AG-3’ |
| Sequence-based reagent | fD2 | Weisburg et al., 1991 | PCR primers | 5’-AGA-GTT-TGA-TCA-TGG-CTC-AG-3’ |
| Sequence-based reagent | rP1 | Weisburg et al., 1991 | PCR primers | 5’-ACG-GTT-ACC-TTG-TTA-CGA-CTT-3’ |
| Commercial assay or kit | QIAquick Gel Extraction Kit | QIAGEN | Cat# 28706 | |
| Commercial assay or kit | ATP Kit SL | BioThema | Cat# 144-041 | |
| Commercial assay or kit | Cell Viability Kit with BD Liquid Counting Beads | BD Biosciences | Cat# 349480 | |
| Commercial assay or kit | Microbial ATP Kit HS | BioThema | Cat# 266-112 | |
| Commercial assay or kit | Intracellular ATP Kit HS | BioThema | Cat# 266-111 | |
| Commercial assay or kit | ReliaPrep RNA Cell Miniprep System | Promega | Cat# Z6011 | |
| Chemical compound, drug | Bacto Yeast Extract | Gibco | Cat# 212750 | |
| Chemical compound, drug | Bacto Tryptone | Gibco | Cat# 211699 | |
| Chemical compound, drug | Agar | Sigma-Aldrich | Cat# 05039-500G | |
| Chemical compound, drug | GoTaq G2 Green Master Mix | Promega | Cat# M782A | |
| Chemical compound, drug | Collagenase I (Col I) | Sigma-Aldrich | Cat# C0130-100MG | |
| Chemical compound, drug | Collagenase IV (Col IV) | Worthington | Cat# LS004189 | |
| Chemical compound, drug | Collagenase D (Col D) | Roche | Cat# 11088858001 | |
| Chemical compound, drug | DNAse I | Roche | Cat# 63792800 | |
| Chemical compound, drug | Adenosine 5′-triphosphate disodium salt hydrate (ATP) | Sigma-Aldrich | Cat# 2383-1G | |
| Chemical compound, drug | Adenosine-5'-(γ-thio)-triphosphate, Tetralithium salt (ATPγs) | Jena Bioscience | Cat# NU-406-50 | |
| Software, algorithm | GraphPad Prism v9.5.1 | Prism GraphPad software | https://www.graphpad.com/ | |
| Software, algorithm | FlowJo v10.8.1 | FlowJo software | https://www.flowjo.com | |
| Software, algorithm | fastqc v0.11.9 | Babraham Bioinformatics, 2024 | http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ | |
| Software, algorithm | fastp v0.19.5 | Chen et al., 2018 | https://github.com/OpenGene/fastp RRID:SCR_016962 | |
| Software, algorithm | STAR v2.7.10a_alpha_220818 | Dobin et al., 2013 | https://github.com/alexdobin/STAR RRID:SCR_004463 | |
| Software, algorithm | subread v2.0.1 | Liao et al., 2014 | https://github.com/ShiLab-Bioinformatics/subread RRID:SCR_009803 | |
| Software, algorithm | R v4.2.2 | The R Project for Statistical Computing | https://cran.r-project.org | |
| Software, algorithm | RStudio v2022.07.2 | RStudio Desktop | https://www.rstudio.com | |
| Software, algorithm | R package ggplot2 v3.4.3 | Wickham, 2016 | https://ggplot2.tidyverse.org | |
| Software, algorithm | R package readxl v1.4.3 | Wickham and Bryan, 2023a | https://CRAN.R-project.org/package=readxl | |
| Software, algorithm | R package ggbreak v0.1.2 | Xu et al., 2021 | https://github.com/YuLab-SMU/ggbreak RRID:SCR_014601 | |
| Software, algorithm | R package rstatix v0.7.2 | Kassambara, 2023 | https://CRAN.R-project.org/package=rstatix | |
| Software, algorithm | R package dplyr v1.0.10 | Wickham et al., 2023b | https://CRAN.R-project.org/package=dplyr | |
| Software, algorithm | R package DESeq2 v1.38.3 | Love et al., 2014 | https://github.com/mikelove/DESeq2 RRID:SCR_015687 | |
| Software, algorithm | R package ggrepel v0.9.3 | Slowikowski et al., 2023 | https://CRAN.R-project.org/package=ggrepel | |
| Software, algorithm | R package vegan v2.6–4 | Oksanen et al., 2022 | https://github.com/vegandevs/vegan RRID:SCR_011950 | |
| Software, algorithm | R package pairwiseAdonis v0.4 | Arbizu, 2023 | https://github.com/pmartinezarbizu/pairwiseAdonis RRID:SCR_001905 | |
| Software, algorithm | R package gprofiler2 v0.2.2 | Kolberg and Raudvere, 2023 | https://cran.r-project.org/web/packages/gprofiler2/index.html | |
| Software, algorithm | R package gplots v3.1.3 | Warnes et al., 2022 | https://CRAN.R-project.org/package=gplots | |
| Software, algorithm | R package viridis v0.6.3 | Ross et al., 2021 | https://sjmgarnier.github.io/viridis/ RRID:SCR_016696 | |
| Other | Fixable Viability Dye eFluor506 | Thermo Fisher Scientific | Cat# 65-0866-18 | (1:600) |
| Other | Nalgene Rapid-Flow PES Filter Units | Thermo Scientific | Cat# 168-0045, 165-0045, 124-0045PK | |
| Other | Vybrant DiI Cell-Labeling Solution | Invitrogen | Cat# V22885 | (1:100) |
| Other | DAPI | Sigma-Aldrich | Cat# D9542-5MG | (1:5000) |
| Other | Hoechst 33342 | Thermo Fisher Scientific | Cat# H3570 | (1:1000) |
| Other | LysoTracker | Invitrogen | Cat# L12492 | (1:1000) |
