Listeria monocytogenes is a major pathogen contributing to foodborne outbreaks with high mortality. Nisin, a natural antimicrobial, has been widely used as a food preservative. However, the mechanisms of L. monocytogenes involved in nisin resistance have not yet to be fully defined. A mariner transposon library was constructed in L. monocytogenes, leading to the identification of 99 genes associated with the innate resistance to nisin via Transposon sequencing (Tn-seq) analysis. To validate the accuracy of the Tn-seq results, we constructed five mutants (ΔyqgS, ΔlafA, ΔvirR, ΔgtcA, and Δlmo1464) in L. monocytogenes. The results revealed that yqgS and lafA, the lipoteichoic acid-related genes, were essential for resistance to nisin, while the gtcA and lmo1464 mutants showed substantially enhanced nisin resistance. Densely wrinkled, collapsed surface and membrane breakdown were shown on ΔyqgS and ΔlafA mutants under nisin treatment. Deletion of yqgS and lafA altered the surface charge, and decreased the resistance to general stress conditions and cell envelope-acting antimicrobials. Furthermore, YqgS and LafA are required for biofilm formation and cell invasion of L. monocytogenes. Collectively, these results reveal novel mechanisms of nisin resistance in L. monocytogenes and may provide unique targets for the development of food-grade inhibitors for nisin-resistant foodborne pathogens. IMPORTANCE Listeria monocytogenes is an opportunistic Gram-positive pathogen responsible for listeriosis, and is widely present in a variety of foods including ready-to-eat foods, meat, and dairy products. Nisin is the only licensed lantibiotic by the FDA for use as a food-grade inhibitor in over 50 countries. A prior study suggests that L. monocytogenes are more resistant than other Gram-positive pathogens in nisin-mediated bactericidal effects. However, the mechanisms of L. monocytogenes involved in nisin resistance have not yet to be fully defined. Here, we used a mariner transposon library to identify nisin-resistance-related genes on a genome-wide scale via transposon sequencing. We found, for the first time, that YqgS and LafA (Lipoteichoic acid-related proteins) are required for resistance to nisin. Subsequently, we investigated the roles of YqgS and LafA in L. monocytogenes stress resistance, antimicrobial resistance, biofilm formation, and virulence in mammalian cells.
Keywords: Listeria monocytogenes; biofilm; lipoteichoic acid; nisin; pathogenicity; transposon sequencing (Tn-seq).