Characterizing the mechanisms of resistance to the novel antimicrobial 4-hydroxy-2-nonenal by the model pathogen Listeria monocytogenes

Abstract

Pathogens encounter numerous antimicrobial responses, including the reactive oxygen species (ROS) burst. ROS-mediated oxidation of host membrane poly-unsaturated fatty acids (PUFAs) generates the toxic ab-carbonyl 4-hydroxy-2-nonenal (4-HNE). Though studied extensively in the context of sterile inflammation, 4-HNE’s role during infection remains limited. Here we found that 4-HNE is generated during bacterial infection and that the intracellular pathogen Listeria monocytogenes induces a specific set of genes in response to 4-HNE exposure. A component of the L. monocytogenes 4-HNE response is the expression of the genes rha1 and rha2 which code for two NADPH-dependent oxidoreductases that collectively counter 4-HNE toxicity by converting 4-HNE to the less toxic product 4-hydroxynonanal (4-HNA). Heterologous expression of rha1/2 in Bacillus subtilis significantly increased bacterial resistance to 4-HNE both in vitro and following phagocytosis by murine macrophages. Our work demonstrates that 4-HNE is a previously unappreciated component of ROS-mediated toxicity and that L. monocytogenes has evolved specific countermeasures to survive within its presence. In addition to the above-mentioned work, we also developed a saturated transposon library in L. monocytogenes that we then used to determine the essential genes of L. monocytogenes using a transposon sequencing (Tn-seq) approach. This methodology could be used in the future to further probe the genetic determinants of L. monocytogenes resistance to 4-HNE.