4-Hydroxy-2-nonenal antimicrobial toxicity is neutralized by an intracellular pathogen

  1. Hannah Tabakh
  2. Adelle P McFarland
  3. Maureen K Thomason
  4. Alex J Pollock
  5. Rochelle C Glover
  6. Shivam A Zaver
  7. Joshua J Woodward  Is a corresponding author
  1. Department of Microbiology, University of Washington, United States
  2. Molecular and Cellular Biology Program, University of Washington, United States
6 figures, 2 tables and 6 additional files

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 levels. Data are normalized 4-HNE/actin levels as percent of 4-HNE/actin in untreated sample. Dot blot image below is representative. (B) 4-HNE accumulation in the spleen after 48 hr murine infection by GFP+ L. monocytogenes assessed by immunohistochemistry analysis with anti-4-HNE antibody. (C) Uninfected spleen with anti-4-HNE antibody. (D) Infected spleen at ×25 magnification with anti-GFP antibody. (E) Infected spleen at ×25 magnification with anti-4-HNE antibody. (F) Infected spleen at ×100 magnification with anti-GFP antibody. (G) Infected spleen at ×100 magnification with anti-4-HNE antibody. Red arrows in D and F indicate L. monocytogenes (GFP) detection in the tissue. Orange arrows in E and G indicate cells with concentrated 4-HNE staining. Antigens were detected with 3,3-diaminobenzidine staining by horseradish peroxidase and cellular nuclei imaged with Hematoxylin counterstain in panels B-G. Data in (A) are in biological quadruplicate. Statistics in (A) are an ordinary one-way ANOVA with a Dunnett’s multiple comparison test against untreated. Error bars are mean ± SD. *p<0.05.

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 antibody. (C) Infected spleen at ×10 magnification with anti-GFP antibody. (D) Infected spleen at ×10 magnification with anti-4-HNE antibody.

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), and Francisella novicida (Fn) following exposure to various concentrations of 4-HNE or mock vehicle (ethanol) in PBS at 37°C for 1 hr. Data are reported as recovered CFU normalized to mock-treated controls. Dashed line is at the limit of detection. (B) Growth of L. monocytogenes in TSB at 37°C with various concentrations of 4-HNE added at time zero. Dashed line at OD600 0.5 (C) Anti-4-HNE dot blot of soluble bacterial lysates from mid-log L. monocytogenes resuspended in PBS and treated with increasing concentrations of 4-HNE for 30 min. Protein levels were normalized (3 µg total protein) and signal quantified by densitometry on a Licor Odyssey Fc. (D) RT-qPCR measurement of expression of the indicated genes from mid-log L. monocytogenes in TSB treated with 640 µM 4-HNE for 20 min. Expression normalized to 16S rRNA levels. (E) Recovered CFU of WT L. monocytogenes following exposure to 4-HNE or mock vehicle (ethanol) in PBS at 37°C for 1 hr, followed by heat shock (50°C) or no heat shock (22°C) treatment for 10 min. Data in figures (A) and (B) are in biological triplicate. Data in (C) and (D) are biological duplicate. Data in (E) are in technical triplicate and representative of at least two independent experiments. Statistics in (E) are an ordinary one-way ANOVA with a Dunnett’s multiple comparison test against each untreated. Error bars are mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. In figure (E), the line is drawn at the median of data.

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 class members are indicated in blue, red, and orange, respectively. (B) RT-qPCR of expression of rha1 and rha2 genes after 20 min treatment of mid-log bacteria in TSB media with 500 µM of selected aldehydes: 4-HNE (4-hydroxy-2-nonenal), 4-HHE (4-hydroxy-2-hexenal), MG (methylglyoxal), PA (propionaldehyde), and MDA (malondialdehyde). (C) RT-qPCR analysis of expression of rha1 and rha1 treated with sublethal levels of diamide (5 mM), heat (50°C), 4-HNE (640 µM), and nitric oxide (1 mM of the NO donor DEA/NO) for 20 min in TSB media. (D) RT-qPCR analysis of expression of rha1 and rha2 at 6 hr post infection in J774 macrophages (mφ). Data in figures (A) and (B) are in biological duplicate. (C) are two independent experiments, with two pooled biological duplicates within each experiment. (D) is in biological triplicate. The bar graphs in (B) (C) and (D) represent the mean of the data. Statistics in (D) are unpaired t-test between the ∆Ct values of broth and macrophage samples. Error bars are mean ± SD. *p<0.05.

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 with sublethal concentrations of listed aldehydes. Performed in technical duplicate. (C) CFU post treatment with a sublethal amount of listed stressors. Performed in technical duplicate.

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) Diameter of zone of clearance by 1M diamide on lawns of WT, ∆rha1∆rha2 and positive control P-spxA1::tn L. monocytogenes on TSA plates as described in Reniere et al., 2016. (D) Accumulation of 4-HNE-adducted proteins in L. monocytogenes exposed to 640 µM 4-HNE for 3 hr in TSB media, assessed by dot blot and normalized to WT. Dot blot images below are representative. (E) Aggregated protein found in the insoluble fraction measured as percent of total protein in WT and ∆rha1∆rha2 L. monocytogenes. Untreated, 4-HNE treatment (640 µL for an hour) and heat shock (56°C for 10 min). (F) CFU/well of WT and ∆rha1∆rha2 L. monocytogenes in recombinant murine IFN-γ (100 ng) activated WT primary murine macrophages. (G) CFU/organ of WT and ∆rha1∆rha2 L. monocytogenes at 48 hr intravenous murine infection. Data in figures (A) and (D) are in technical triplicate, representative of at least three independent experiments. Data in (C) are two independent experiments, with two pooled biological duplicates within each experiment. Data in (B), (E), (F) and (G) are biological triplicate. Statistics in (A) are unpaired t-tests between WT and mutant L. monocytogenes competition pairs. Statistics in (C) are unpaired t-tests between WT and mutant L. monocytogenes. Statistics in (D) and (E) are an ordinary one-way ANOVA with a Dunnett’s multiple comparison test against WT (in D) or untreated sample (in E). Statistics in (F) are unpaired t-tests comparing WT and ∆rha1∆rha2 L. monocytogenes CFU at hour two post infection. Statistics in (G) are unpaired t-tests comparing WT and ∆rha1∆rha2 L. monocytogenes CFU within each organ. Error bars are mean ± SD. *p<0.05; **p<0.01; ***p<0.001. In figures (A) and (G), the line is drawn at the median of data.

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. 

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 (blue) and mutant (orange) variants of Rha1 and Rha2 in the presence of 4-HNE. (C) Rate of NADPH oxidation (200 µM) by human enzyme AKR1C1 in the presence of 4-HNE. (D) NADPH oxidation rate by Rha1 and Rha2 in the presence of 640 µM final concentration of various aldehydes. (E) TLC plates showing the migration of reaction contents of Lane 1: 4-HNE, Lane 2: 1,4-DHN, and Lanes 3–6: indicated enzymes with 4-HNE in the absence and presence of NADPH after 1 hr of reaction at room temperature. 4-HNE -- black arrow, 4-HNA -- red arrow, 1,4-DHN -- blue arrow. (F) Diagram of 4-HNE to 4-HNA conversion. Data in (B), (C), (D) and (E) are representative of at least three independent experiments.

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 zero. Dotted line represents OD600 0.5. (B) Survival of B. subtilis WT and MUT in PBS at 37°C with 160 µM 4-HNE for 1 hr. (C) 4-HNE conjugates from B. subtilis WT and MUT soluble cell lysates (3 µg total protein) 3 hr after 4-HNE treatment as assessed by dot blot and quantified by densitometry on a Licor Odyssey Fc. Dot blots below are representative. (D) B. subtilis WT and MUT survival following phagocytosis by Interferon gamma-activated primary WT or phagosomal oxidase-deficient bone-marrow-derived macrophages (WT or gp91phox-/- pBMMs). All experiments were performed in biological triplicate. Statistics in (A) are unpaired t-tests comparing the hours to OD600 0.5 between WT and MUT B. subtilis pHT01::rha1/2. Statistics in (B), (C) and (D) are unpaired t-tests comparing WT and MUT B. subtilis pHT01::rha1/2. Error bars are mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

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 squares: 1280 µM). Data performed in technical duplicate and representative of at least three independent experiments. (B) Growth curves of B. subtilis::pHT01 expressing various rha1 and rha2 constructs in TSB after exposure to various concentrations of 4-HNE. Dotted line represents OD600 0.5. Performed in biological triplicate. (C) 4-HNE dot blots of B. subtilis::pHT01 expressing various rha1 and rha2 constructs after 3 hr of exposure to 640 µM 4-HNE in TSB. Performed in biological triplicate. Statistics in (B) is an unpaired t-test comparing the hours to OD600 0.5 between B. subtilis pHT01::rha1-WT and pHT01::rha1-N47A. Error bars are mean ± SD. *p<0.05.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Mus musculus)J774A.1PMID:1612739Immortalized murine macrophages
Cell line (Mus musculus)TIB73ATCC: BNL CL.2Immortalized murine hepatocytes
Strain, strain background (Mus musculus)C57BL/6JJackson Laboratories: 000664
Strain, strain background (Mus musculus)gp91phox- (C57BL/6J backcross)Jackson Laboratories: 002365
Strain, strain background (Escherichia coli)DH10bInvitrogen: 10536193
Strain, strain background (Escherichia coli)SM10DOI: 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)10403SPMID:2125302WT
Strain, strain background (Bacillus subtilis)168PMID:18723616WT
Strain, strain background (Francisella novicida)U112PMID:17550600WT
Strain, strain background (Pseudomonas aeruginosa)PAO1PMID:20023018WT
Strain, strain background (Staphylococcus aureus)NewmanPMID:17951380WT
Strain, strain background (Enterococcus faecalis)OG1RFPMID:18611278WT
Strain, strain background (Listeria monocytogenes)DP-L3903PMID:11500481Antibiotic marked competition strain
Strain, strain background (Listeria monocytogenes)DP-L4056PMID:12107135Phage-cured 10403S strain for use with pPL1 integration plasmid
Genetic reagentGenetically modified bacterial strains used in this workThis paperSupplementary file 1
recombinant DNA reagentpPL1077This studypPL1-GFP integration plasmid(Gift from Peter Lauer; Berkeley, CA)
Recombinant DNA reagentpKSV7PMID:8388529Shuttle vector for L. monocytogenes gene disruption
Recombinant DNA reagentpET20bNovagen: 69739C-terminus 6xHis tagged E. coli T7 expression vector
Recombinant DNA reagentpET28bNovagen: 69865N-terminus 6xHis tagged E. coli T7 expression vector
Recombinant DNA reagentpPL2-PspacPMID:25583510Integrative L. monocytogenes plasmid pPL2 engineered with constitutive Pspac promoter
Recombinant DNA reagentpHT01PMID:17624574B. subtilis expression vector
Recombinant DNA reagentsPlasmids generated and used in this workThis paperSupplementary file 2
Gene (Listeria monocytogenes)lmo0103 (LMRG_02352)GenBank: Gene ID 12552319Gene rha1
Gene (Listeria monocytogenes)lmo0613 (LMRG_00296)GenBank: Gene ID 12552833Gene rha2
Gene (Homo sapiens)akr1c1GenBank: Gene ID 1645Gene akr1c1
Gene (Arabidopsis thaliana)p1-zcrGenBank: Gene ID 831560Gene p1-zcr
Sequence-based reagentsRT-qPCR primersThis paper(See supplementary file 3)
Sequence-based reagentsCloning primersThis paper(See supplementary file 3)
Sequence-based reagentsGene coding sequences for protein expressionThis paper(See supplementary file 4)
Chemical, compound, drug4-HNECayman Chemical: 3210064 mM 4-HNE in absolute ethanol
AntibodyAnti-4-HNE adductAbcam: ab46545Rabbit IgG polyclonal antibody(1:200)
AntibodyAnti-actinAbcam: ab8226Mouse IgG monoclonal antibody(1:1000)
AntibodyAnti-rabbit secondary antibodyLicor: 926–32211Goat IgG monoclonal antibody(1:8000)
AntibodyAnti-mouse secondary antibodyLicor: 926–68072Donkey IgG monoclonal antibody(1:8000)
AntibodyAnti-GFP primary antibodyInvitrogen: MA5-15256Mouse IgG monoclonal antibody(1:500)
AntibodyIsotype control primary antibody, histologyR and D Systems: AB-105-CNormal Rabbit IgG(1:1000)
Commercial assay, kitOvation Complete Prokaryotic RNA-Seq SystemNugen: 0363–32RNA-seq processing kit
Software, algorithmRockhoppercs.wellesley.edu/~btjaden/Rockhopper/RNA-seq data analysis software
Author response table 1
OrganismRha1 identity (%)Rha2 identity (%)
Pa2931
SAAbsent32
Ef5971
EcAbsent34
Fn2433
Bs5829

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
Supplementary file 1

Bacterial strains used in this study.

https://cdn.elifesciences.org/articles/59295/elife-59295-supp1-v2.xlsx
Supplementary file 2

Plasmids used in this study.

https://cdn.elifesciences.org/articles/59295/elife-59295-supp2-v2.xlsx
Supplementary file 3

Primers used in this study.

https://cdn.elifesciences.org/articles/59295/elife-59295-supp3-v2.xlsx
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
Transparent reporting form
https://cdn.elifesciences.org/articles/59295/elife-59295-transrepform-v2.pdf

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  1. Hannah Tabakh
  2. Adelle P McFarland
  3. Maureen K Thomason
  4. Alex J Pollock
  5. Rochelle C Glover
  6. Shivam A Zaver
  7. Joshua J Woodward
(2021)
4-Hydroxy-2-nonenal antimicrobial toxicity is neutralized by an intracellular pathogen
eLife 10:e59295.
https://doi.org/10.7554/eLife.59295