Host-induced spermidine production in motile Pseudomonas aeruginosa triggers phagocytic uptake
- Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Germany
- Department of Chemical Biology, Helmholtz Centre for Infection Research, Germany
- Department of Cell Biology, Helmholtz Centre for Infection Research, Germany
- Central Facility for Microscopy, Helmholtz Centre for Infection Research, Germany
- Department of Molecular Bacteriology, Twincore, Germany
- Department of Clinical Microbiology, Rigshospitalet, Denmark
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Germany
Figures
Figure 1
Phenotypic characterization of the P. aeruginosa strains.
(A) Schematic depiction of the mono-flagellated, fully motile PA14 wild-type phenotype and three isogenic mutants which are non-motile due to the lack of flagella (ΔfliC), non-assembled flagellin (Δf…
-
Figure 1—source data 1
Bacterial strains and plasmids used in this study.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig1-data1-v2.xlsx
-
Figure 1—source data 2
Scanning Electron microscopy.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig1-data2-v2.pdf
-
Figure 1—source data 3
Motility Plates.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig1-data3-v2.pdf
Figure 2 with 2 supplements
Reduced macrophage uptake of non-flagellated and non-motile P. aeruginosa strains.
Phagocytic uptake 1 hr post infection of PA14 and the three motility variants into bone-marrow-derived macrophages (BMDMs) (A) and RAW264.7 cells (B) using a multiplicity of infection (MOI) of 1. …
-
Figure 2—source data 1
Negative Stain.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig2-data1-v2.pdf
Figure 2—figure supplement 1
Phagocytosis and adherence of P. aeruginosa motility variants.
(A) Influence of a centrifugation step on the bacterial uptake of non-motile ΔflgK by J774 macrophages. Mean ± standard deviation of three biological replicates is displayed (B) Phagocytic uptake of …
Figure 2—figure supplement 2
FliC complementation restores the wild-type phenotype.
(A) Representative pictures of the individual P. aeruginosa variants analyzed by scanning electron microscopy. (B) Swimming motility assessed on semisolid agar containing 500 µg/ml kanamycin after …
Figure 3 with 4 supplements
Dual-sequencing approach.
(A) Experimental set-up to record the transcriptional profiles of infected RAW264.7 macrophages, P. aeruginosa upon host-cell contact, and P. aeruginosa grown in rich medium as a planktonic culture. …
-
Figure 3—source data 1
Summary of the 20 differentially expressed genes that are shared by the motility mutants in comparison to PA14 Wt.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig3-data1-v2.xlsx
-
Figure 3—source data 2
Significantly enriched host pathways.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig3-data2-v2.csv
Figure 3—figure supplement 1
Venn Diagrams to visualize the dual-seq transcriptional responses.
(A) Genes that were differentially expressed under LB growth conditions in the three motility mutants as compared to PA14. (B) Visualization of differentially regulated host genes in RAW264.7 …
Figure 3—figure supplement 2
Lactate Dehydrogenase (LDH) assay of infected RAW264.7 macrophages.
RAW macrophages were infected with 5 × 105 – MOI 1 for 3 hr without gentamycin treatment to simulate the dual-sequencing experiment. PBS served as the negative and 10% (v/v) Triton-X100 as killing …
Figure 3—figure supplement 3
Pathogen-to-host read ratio.
The read ratio between read counts of bacteria and host cells was analyzed. Mapped paired end reads of P. aeruginosa strains were normalized according their exact infection dose (approximately MOI …
Figure 3—figure supplement 4
Functional GO term enrichment.
Enriched gene functions (GO biological process) of the individual mutant strains in comparison to PA14 under infection conditions (see Figure 3C). Functional enrichment of genes that were …
-
Figure 3—figure supplement 4—source data 1
Enriched functions in the Pseudomonas variants.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig3-figsupp4-data1-v2.csv
Figure 4 with 1 supplement
Motility mutant specific transcriptional responses in the presence of macrophages.
Genes that were differentially expressed (log2FC, glmTreat function, R package edgeR) in all three flagella mutants as compared to PA14 (n = 53 genes) are listed. The color code depicts the log2FC …
-
Figure 4—source data 1
Bacterial gene regulation.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig4-data1-v2.csv
-
Figure 4—source data 2
Positive interspecies correlation of 53 PA14 genes and their association with 74 host PIP3 genes.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig4-data2-v2.xlsx
-
Figure 4—source data 3
Negative interspecies correlation of 53 PA14 genes and their association with 74 host PIP3 genes.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig4-data3-v2.xlsx
-
Figure 4—source data 4
Differentially expressed genes between the fliC mutant and PA14 wild type after a medium switch from LB to DMEM medium containing FCS.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig4-data4-v2.xlsx
Figure 4—figure supplement 1
Cytokine measurement in the supernatant of infected macrophages using ELISA.
TNF-α measurements of RAW264.7 (A) and BMDM cells (B) after 6 hr infection with P. aeruginosa at an MOI of 1. IL-6 measurements of RAW264.7 (C) and BMDM cells (D) after 6 hr infection with P. …
Figure 5
Non-motile variants of P. aeruginosa elicit a reduced activation of the PIP3 pathway in macrophages.
(A) Dual-seq analysis revealed that the pathways leading to the signaling molecules PI(3,4)P2 and PI(3,4,5)P3 (in red), and the activation of the calcium-dependent cell rearrangements are expressed …
-
Figure 5—source data 1
Enrichment of calcium and PIP3-related functions of a downregulated (log2FC < 0) subset of Top 1000 genes from ΔfliC, ΔflgK and ΔmotABCD mutants.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig5-data1-v2.xlsx
Figure 6 with 1 supplement
Spermidine enhances phagocytic uptake.
(A) Spermidine was externally added at the indicated concentrations to the ΔfliC mutant. Using MOI of 1, the phagocytic uptake in RAW264.7 macrophages was examined 1 hr post infection. Mean ± standar…
-
Figure 6—source data 1
Spermidine induced gene induction.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig6-data1-v2.csv
-
Figure 6—source data 2
Shared enriched functions of spermidine-treated and PA14-infected macrophages.
- https://cdn.elifesciences.org/articles/55744/elife-55744-fig6-data2-v2.xlsx
Figure 6—figure supplement 1
Effect of spermidine on phagocytic uptake.
(A) Effect of spermidine on the phagocytic uptake of PA14 in RAW264.7 macrophages using an MOI of 1. Mean ± standard deviation of three biological replicates is displayed. (B) Effect of 10 µM …
Figure 7 with 2 supplements
Spermidine and norspermidine restore phagocytic uptake of a spermidine knock-out mutant.
Spermidine was externally added at the indicated concentrations to the ΔPA14_63120 mutant (blue). Using an MOI 1, the phagocytic uptake into RAW264.7 macrophages 1 hr post infection was examined and …
Figure 7—figure supplement 1
Quantification of spermidine using LC-MS.
All strains were grown in BM2 media containing glucose as carbon source. Membrane-bound spermidine was extracted from PA14 and its motility variants, derivatized with phenylisothiocyanate and …
Figure 7—figure supplement 2
Effect of norspermidine on phagocytic uptake.
(A) Effect of norspermidine on the phagocytic uptake of PA14 in RAW264.7 macrophages using an MOI of 1. Mean ± standard deviation of three biological replicates is displayed. (B) Effect of 10 µM …
Figure 8
Flagellum-dependent Pseudomonas–macrophage interaction.
Upon host contact, changes in flagellar activity (possibly sensed by PmrAB and PhoPQ) trigger a transcriptional program, which results in the synthesis of membrane-associated spermidine and the …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | Bacto-Agar | BD | 214010 | - |
| Chemical compound, drug | Gentamycin | Sigma | G1397-10ml | - |
| Chemical compound, drug | PtdIns(3,4,5)P3 | Echelon | P-3916 | - |
| Chemical compound, drug | Shuttle PIPTM Carrier 2 | Echelon | P-9C2 | - |
| Chemical compound, drug | DMEM | Gibco | 31885–023 | - |
| Chemical compound, drug | RPMI | Gibco | 21875–034 | - |
| Chemical compound, drug | FCS | Biowest | S1810-500 | - |
| Chemical compound, drug | BSA | Sigma | A9418-50 | - |
| Chemical compound, drug | HEPES | Gibco | 15630–056 | - |
| Chemical compound, drug | Cytochalasin D | Sigma | C2618-200UL | - |
| Chemical compound, drug | Spermidine | Sigma | S0266-25G | - |
| Chemical compound, drug | Norspermidine | Sigma | I1006A-100G-A | - |
| Chemical compound, drug | TritonX-100 | BioRad | #161–0407 | - |
| Chemical compound, drug | RNAprotect Bacteria Reagent | Qiagen | #76506 | - |
| Chemical compound, drug | Pyridine, HPLC Grade, 99.5+% | Alfa Aesar | 110-86-1 | - |
| Chemical compound, drug | Phenylisothiocyanate (PITC) | Sigma | 78780 | - |
| Antibody | Rabbit anti- AKT | Cell Signaling Technology | #9272 | 1:1000 |
| Antibody | Rabbit anti-p-AKT (Ser473) | Cell Signaling Technology | #9271 | 1:1000 |
| Antibody | Mouse anti-GAPDH | Calbiochem | CB1001 | 1:10000 |
| Antibody | Goat anti-mouse IgG+IgM (H+L) | Dianova | 115-035-068 | 1:10000 |
| Antibody | Goat anti-rabbit IgG (H+L) | Dianova | 111-035-045 | 1:10000 |
| Commercial assay, kit | NEBNext Single Cell/low input Input RNA Kit | NEB | E6240S | - |
| Commercial assay, kit | Ribo-Zero rRNA removal Kit | Illumina | - | - |
| Commercial assay, kit | NEBNext Ultra II Directional RNA Library Prep Kit | NEB | E7760L | - |
| Commercial assay, kit | ELISA MAX Standard Set Mouse TNF-α | BioLegend | #430901 | - |
| Commercial assay, kit | ELISA MAX Standard Set Mouse IL-6 ELISA MAX Standard Set Mouse IL-6 ELISA MAX Standard Set Mouse IL-6 | BioLegend | #431301 | - |
| Cell line (M. musculus) | RAW264.7 macrophages | - | RRID:CVCL_0493 | - |
| Cell line (M. musculus) | J774 cells | - | RRID:CVCL_0358 | |
| Cell line (M. musculus) | BMDMs C57BL/6 | Own breeding | N/A | - |
| Cell line (M. musculus) | L929 | - | RRID:CVCL_0462 | - |
| Strain, strain background (P. aeruginosa) | PA14 wild-type | Amiel et al., 2010 | N/A | - |
| Strain, strain background (P. aeruginosa) | ΔfliC | Amiel et al., 2010 | N/A | - |
| Strain, strain background (P. aeruginosa) | ΔflgK | Amiel et al., 2010 | N/A | - |
| Strain, strain background (P. aeruginosa) | ΔmotABCD | Amiel et al., 2010 | N/A | - |
| Strain, strain background (P. aeruginosa) | ΔPA14_63120 | This study | N/A | - |
| Strain, strain background (P. aeruginosa) wild-type | CH2677 | Hornischer et al., 2019 | N/A | Clinical Isolate |
| Strain, strain background (P. aeruginosa) wild-type | CH3290 | Hornischer et al., 2019 | N/A | Clinical Isolate |
| Strain, strain background (P. aeruginosa) | CH3797 | Hornischer et al., 2019 | N/A | Clinical Isolate |
| Strain, strain background (P. aeruginosa) | CH4035 | Hornischer et al., 2019 | N/A | Clinical Isolate |
| Strain, strain background (P. aeruginosa) | CH4433 | Hornischer et al., 2019 | N/A | Clinical Isolate |
| Strain, strain background (P. aeruginosa) | CH4438 | Hornischer et al., 2019 | N/A | Clinical Isolate |
| Strain, strain background (P. aeruginosa) | CH5334 | Hornischer et al., 2019 | N/A | Clinical Isolate |
| Strain, strain background (P. aeruginosa) | CH5528 | Hornischer et al., 2019 | N/A | Clinical Isolate |
| Strain, strain background (M. musculus) | C57BL/6 | Own Breeding | N/A | - |
| Recombinant DNA reagent | pEX18-Gm | Lab stock | N/A | Suicide vector |
| Software, algorithm | ImageJ | - | RRID:SCR_003070 | v1.52p |
| Software, algorithm | Prism | Graphpad | N/A | v5.01 |
| Software, algorithm | Zeiss SEM Smart | Zeiss | N/A | v5.05 |
| Software, algorithm | ITEM Build 1210 | Olympus Soft Imaging | N/A | - |
| Software, algorithm | Skyline | MacCoss Laboratory | N/A | v18.305 |
| Software, algorithm | R Project | - | RRID:SCR_001905 | v3.6.1 |
| Software, algorithm | Analyst | ABSciex | N/A | v1.6.2 |
| Software, algorithm | Fastqc | N/A | RRID:SCR_014583 | v0.11.4 |
| Software, algorithm | tophat2 | N/A | RRID:SCR_013035 | v2.0.12 |
| Software, algorithm | Bowtie2 | N/A | RRID:SCR_005476 | v2.3.4.1 |
| Software, algorithm | Samtools | N/A | RRID:SCR_002105 | v0.1.19.0 |
| Software, algorithm | bedtools | N/A | RRID:SCR_006646 | v2.29.0 |
| Software, algorithm | DAVID (https://david.ncifcrf.gov/) | N/A | RRID:SCR_001881 | v6.7 |
| Software, algorithm | R library Rsubread | N/A | RRID:SCR_016945 | v1.34.7 |
| Software, algorithm | R library edgeR | N/A | RRID:SCR_012802 | v3.24.3 |
| Software, algorithm | R library eulerr | Larsson, 2018 | N/A | v6.0.0 |
| Software, algorithm | R library superheat | Barter and Yu, 2018 | N/A | v0.1.0 |
| Software, algorithm | R library pheatmap | N/A | RRID:SCR_016418 | v1.0.12 |
| Software, algorithm | R library ggplot2 | N/A | RRID:SCR_014601 | v3.1.1 |
| Software, algorithm | R library RColorBrewer | N/A | RRID:SCR_016697 | v1.1–2 |
| Other | Reference genome M. musculus | GRCm38/mm10 | N/A | - |
| Other | Reference genome P. aeruginosa UCBPP-PA14 | NC_008463.1 | N/A | - |
