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

Abstract

Pathogens encounter numerous antimicrobial responses during infection, including the reactive oxygen species (ROS) burst. ROS-mediated oxidation of host membrane poly-unsaturated fatty acids (PUFAs) generates the toxic alpha-beta carbonyl 4-hydroxy-2-nonenal (4-HNE). Though studied extensively in the context of sterile inflammation, research into 4-HNE's role during infection remains limited. Here we found that 4-HNE is generated during bacterial infection, that it impacts growth and survival in a range of bacteria, and that the intracellular pathogen Listeria monocytogenes induces many genes in response to 4-HNE exposure. A component of the L. monocytogenes 4-HNE response is the expression of the genes lmo0103 and lmo0613, deemed rha1 and rha2 (reductase of host alkenals), respectively, which code for two NADPH-dependent oxidoreductases that convert 4-HNE to the product 4-hydroxynonanal (4-HNA). Loss of these genes had no impact on L. monocytogenes bacterial burdens during murine or tissue culture infection. However, heterologous expression of rha1/2 in Bacillus subtilis significantly increased bacterial resistance to 4-HNE in vitro and promoted bacterial survival following phagocytosis by murine macrophages in an ROS dependent manner. Thus, Rha1 and Rha2 are not necessary for 4-HNE resistance in L. monocytogenes but are sufficient to confer resistance to an otherwise sensitive organism in vitro and in host cells. Our work demonstrates that 4-HNE is a previously unappreciated component of ROS-mediated toxicity encountered by bacteria within eukaryotic hosts.

The aldehyde hypothesis: metabolic intermediates as antimicrobial effectors

“…Because this phenotype is rescued by constitutive activation of PrfA, a bacterial transcription factor activated upon entry into host cell cytosol, the authors speculate that host-derived MG is a cue that bacteria sense to activate transcription of virulence genes. In addition, Woodward and co-workers [ 45 ] proposed L. monocytogenes encodes genes to deal with 4-HNE generated by ROS during infections. In this study, the authors looked for genes that were induced in the presence of 4-HNE, with the hypothesis that these genes encode detoxifying products of this lipid aldehyde encountered in animal tissues.…”

Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,β-Unsaturated Aldehydes in Listeria monocytogenes

“…S3). A fourth α,β-unsaturated aldehyde that was tested is 4-HNE, which is a degradation product of polyunsaturated fatty acids that is formed during the oxidative burst as part of the cellular immunity ( 34 , 35 ). Since the compound is bactericidal at low concentrations and may play a role in pathogen elimination during infection, we tested its activity in a killing assay rather than in a growth inhibition assay ( Fig.…”

“…The α,β-unsaturated aldehyde group of 4-HNE easily undergoes a Michael-type addition reaction with thiol and amino groups of proteins to generate protein adducts, and this reactivity is held responsible for its antibacterial activity ( 34 , 37 , 38 ). A recent study found that two NADPH-dependent oxidoreductases, namely, the flavin-dependent enone reductase Rha1 and the alcohol/quinone reductase Rha2, play pivotal roles in the resistance of L. monocytogenes against 4-HNE ( 35 ). Recombinant Rha1 and Rha2 reduced the C=C double bond of 4-HNE, generating 4-hydroxynonanal in a NADPH-dependent manner.…”

“…Recombinant Rha1 and Rha2 reduced the C=C double bond of 4-HNE, generating 4-hydroxynonanal in a NADPH-dependent manner. However, against 4-hydroxy-2-hexenal, Rha1 and Rha2 exhibited no activity and modest activity, respectively ( 35 ), indicating the narrow substrate specificities of the two enzymes. The expression of both Rha1 and Rha2 was highly induced (238-fold and 180-fold, respectively) in response to 640 μM 4-HNE, as revealed via a transcriptome analysis.…”

“…Since our work shows that the absence of YhfK completely abolished the production of 3-phenylpropanal from t-CIN, it can be concluded that none of Rha1, Rha2, or any other ene reductases reduce the double bond of t-CIN in L. monocytogenes . Although a role in virulence could not be demonstrated, Rha1 and Rha2 were shown to contribute to the tolerance of L. monocytogenes to 4-HNE in vitro , and the heterologous expression of the genes promoted the survival of B. subtilis , following phagocytosis by macrophages ( 35 ). Since our present work shows that YhfK also protects L. monocytogenes against 4-HNE, it would be worthwhile to investigate whether this enzyme also contributes to pathogenicity.…”