4-Hydroxy-2-nonenal antimicrobial toxicity is neutralized by an intracellular pathogen
- Department of Microbiology, University of Washington, United States
- Molecular and Cellular Biology Program, University of Washington, United States
Figures
Figure 1 with 1 supplement
4-HNE accumulates during intracellular bacterial infection by L. monocytogenes.
(A) 4-HNE accumulation in TIB73 murine hepatocytes during intracellular L. monocytogenes infection. 4-HNE adduct accumulation was assessed by dot blot of whole cell lysates normalized to actin …
Figure 1—figure supplement 1
4-HNE does not accumulate in the liver during infection by L. monocytogenes.
(A) 4-HNE accumulation in the spleen after 48 hr murine infection by GFP+ L. monocytogenes assessed by immunohistochemistry analysis with anti-4-HNE antibody. (B) Uninfected spleen with anti-4-HNE …
Figure 2
4-HNE is a bactericidal, cell-permeable and protein damaging molecule.
(A) Survival of mid-log (0.4–0.8 OD600) Listeria monocytogenes (Lm), Enterococcus faecalis (Ef), Pseudomonas aeruginosa (Pa), Escherichia coli (Ec), Staphylococcus aureus (Sa), Bacillus subtilis (Bs)…
Figure 3 with 2 supplements
4-HNE exposure induces resistance genes in L. monocytogenes.
(A) Global gene expression of mid-log L. monocytogenes in TSB treated with 640 µM 4-HNE or ethanol control for 20 min. RPKM: reads per kilobase million. Genes of interest rha1, rha2, and heat shock …
Figure 3—figure supplement 1
Global gene expression of mid-log L. monocytogenes in TSB treated with ethanol mock control (panel 1) or 640 µM 4-HNE (panel 1) for 20 min.
RPKM: reads per kilobase million.
Figure 3—figure supplement 2
Various aldehydes and chemical stresses and impacts on L. monocytogenes survival.
(A) Structures of the aldehydes used in the experiment. 4-HNE: 4-hydroxy-2-nonenal, 4-HHE: 4-hydroxy-2-hexenal, MDA: malondialdehyde, MG: methylglyoxal, PA: propionaldehyde. (B) CFU post treatment …
Figure 4 with 1 supplement
L. monocytogenes ∆rha1∆rha2 has reduced ability to survive 4-HNE toxicity.
(A) Competitive index of WT and mutant L. monocytogenes in PBS treated with 640 µM 4-HNE at 37°C for 1 hr. (B) CFU of WT and ∆rha1∆rha2 L. monocytogenes in TSB exposed to 58°C or 37°C for 15 min. (C)…
Figure 4—figure supplement 1
Impact of ∆rha1 and ∆rha2 on L. monocytogenes survival in PBS.
(A) Competitive index of 1 hr mock -treated (ethanol) L. monocytogenes in PBS. The line is drawn at the median of data.
Figure 5
Recombinant Rha1 and Rha2 metabolize 4-HNE to 4-HNA.
(A) Phyre2 structural homology models predict that asparagine-47 interacts with the FMN in Rha1 (left) and tyrosine-195 coordinates NADPH in Rha2 (right). (B) Rates of NADPH oxidation (200 µM) by WT …
Figure 6 with 1 supplement
Ectopic expression or Rha1 and Rha2 confers 4-HNE resistance to sensitive bacteria.
(A) Growth of B. subtilis expressing either the WT or MUT (catalytically dead mutant) versions of rha1 and rha2 in TSB at 37°C in the presence of the indicated concentrations of 4-HNE added at time …
Figure 6—figure supplement 1
Impacts of 4-HNE, Rha1, and Rha2 on 4-HNE growth delay and adduct accumulation in a sensitive bacteria.
(A) L. monocytogenes (Lm) and B. subtilis (Bs) growth curves in TSB after exposure to various concentrations of 4-HNE. (green circles: 0 µM, blue triangles: 320 µM, red triangles: 640 µM, light blue …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Mus musculus) | J774A.1 | PMID:1612739 | Immortalized murine macrophages | |
| Cell line (Mus musculus) | TIB73 | ATCC: BNL CL.2 | Immortalized murine hepatocytes | |
| Strain, strain background (Mus musculus) | C57BL/6J | Jackson Laboratories: 000664 | ||
| Strain, strain background (Mus musculus) | gp91phox- (C57BL/6J backcross) | Jackson Laboratories: 002365 | ||
| Strain, strain background (Escherichia coli) | DH10b | Invitrogen: 10536193 | ||
| Strain, strain background (Escherichia coli) | SM10 | DOI: https://doi.org/10.1038/nbt1183-784 | ||
| Strain, strain background (Escherichia coli) | BL21 (DE3) | EMD Millipore: 69450 | ||
| Strain, strain background (Escherichia coli) | Rosetta (DE3) | EMD Millipore: 70954 | ||
| Strain, strain background (Listeria monocytogenes) | 10403S | PMID:2125302 | WT | |
| Strain, strain background (Bacillus subtilis) | 168 | PMID:18723616 | WT | |
| Strain, strain background (Francisella novicida) | U112 | PMID:17550600 | WT | |
| Strain, strain background (Pseudomonas aeruginosa) | PAO1 | PMID:20023018 | WT | |
| Strain, strain background (Staphylococcus aureus) | Newman | PMID:17951380 | WT | |
| Strain, strain background (Enterococcus faecalis) | OG1RF | PMID:18611278 | WT | |
| Strain, strain background (Listeria monocytogenes) | DP-L3903 | PMID:11500481 | Antibiotic marked competition strain | |
| Strain, strain background (Listeria monocytogenes) | DP-L4056 | PMID:12107135 | Phage-cured 10403S strain for use with pPL1 integration plasmid | |
| Genetic reagent | Genetically modified bacterial strains used in this work | This paper | Supplementary file 1 | |
| recombinant DNA reagent | pPL1077 | This study | pPL1-GFP integration plasmid | (Gift from Peter Lauer; Berkeley, CA) |
| Recombinant DNA reagent | pKSV7 | PMID:8388529 | Shuttle vector for L. monocytogenes gene disruption | |
| Recombinant DNA reagent | pET20b | Novagen: 69739 | C-terminus 6xHis tagged E. coli T7 expression vector | |
| Recombinant DNA reagent | pET28b | Novagen: 69865 | N-terminus 6xHis tagged E. coli T7 expression vector | |
| Recombinant DNA reagent | pPL2-Pspac | PMID:25583510 | Integrative L. monocytogenes plasmid pPL2 engineered with constitutive Pspac promoter | |
| Recombinant DNA reagent | pHT01 | PMID:17624574 | B. subtilis expression vector | |
| Recombinant DNA reagents | Plasmids generated and used in this work | This paper | Supplementary file 2 | |
| Gene (Listeria monocytogenes) | lmo0103 (LMRG_02352) | GenBank: Gene ID 12552319 | Gene rha1 | |
| Gene (Listeria monocytogenes) | lmo0613 (LMRG_00296) | GenBank: Gene ID 12552833 | Gene rha2 | |
| Gene (Homo sapiens) | akr1c1 | GenBank: Gene ID 1645 | Gene akr1c1 | |
| Gene (Arabidopsis thaliana) | p1-zcr | GenBank: Gene ID 831560 | Gene p1-zcr | |
| Sequence-based reagents | RT-qPCR primers | This paper | (See supplementary file 3) | |
| Sequence-based reagents | Cloning primers | This paper | (See supplementary file 3) | |
| Sequence-based reagents | Gene coding sequences for protein expression | This paper | (See supplementary file 4) | |
| Chemical, compound, drug | 4-HNE | Cayman Chemical: 32100 | 64 mM 4-HNE in absolute ethanol | |
| Antibody | Anti-4-HNE adduct | Abcam: ab46545 | Rabbit IgG polyclonal antibody | (1:200) |
| Antibody | Anti-actin | Abcam: ab8226 | Mouse IgG monoclonal antibody | (1:1000) |
| Antibody | Anti-rabbit secondary antibody | Licor: 926–32211 | Goat IgG monoclonal antibody | (1:8000) |
| Antibody | Anti-mouse secondary antibody | Licor: 926–68072 | Donkey IgG monoclonal antibody | (1:8000) |
| Antibody | Anti-GFP primary antibody | Invitrogen: MA5-15256 | Mouse IgG monoclonal antibody | (1:500) |
| Antibody | Isotype control primary antibody, histology | R and D Systems: AB-105-C | Normal Rabbit IgG | (1:1000) |
| Commercial assay, kit | Ovation Complete Prokaryotic RNA-Seq System | Nugen: 0363–32 | RNA-seq processing kit | |
| Software, algorithm | Rockhopper | cs.wellesley.edu/~btjaden/Rockhopper/ | RNA-seq data analysis software |
Author response table 1
| Organism | Rha1 identity (%) | Rha2 identity (%) |
|---|---|---|
| Pa | 29 | 31 |
| SA | Absent | 32 |
| Ef | 59 | 71 |
| Ec | Absent | 34 |
| Fn | 24 | 33 |
| Bs | 58 | 29 |
Additional files
-
Source data 1
Global gene expression of mid-log L. monocytogenes treated with 640 µM 4-HNE or ethanol control for 20 min.
- https://cdn.elifesciences.org/articles/59295/elife-59295-data1-v2.docx
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Supplementary file 1
Bacterial strains used in this study.
- https://cdn.elifesciences.org/articles/59295/elife-59295-supp1-v2.xlsx
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Supplementary file 2
Plasmids used in this study.
- https://cdn.elifesciences.org/articles/59295/elife-59295-supp2-v2.xlsx
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Supplementary file 3
Primers used in this study.
- https://cdn.elifesciences.org/articles/59295/elife-59295-supp3-v2.xlsx
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Supplementary file 4
DNA coding sequences used for protein expression and trans complementation in this study.
- https://cdn.elifesciences.org/articles/59295/elife-59295-supp4-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/59295/elife-59295-transrepform-v2.pdf
