Department of Food Science and Nutrition, Zhejiang Universitygrid.13402.34, Hangzhou, China.
College of Agriculture and Forestry, Linyi University, Linyi, China.
Microbiol Spectr. 2022 Feb 23;10(1):e0209521. doi: 10.1128/spectrum.02095-21.
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 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 (Δ, Δ, Δ, Δ, and Δ) in L. monocytogenes. The results revealed that and , the lipoteichoic acid-related genes, were essential for resistance to nisin, while the and mutants showed substantially enhanced nisin resistance. Densely wrinkled, collapsed surface and membrane breakdown were shown on Δ and Δ mutants under nisin treatment. Deletion of and 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. 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.
单增李斯特菌是一种主要的食源性病原体，可导致高死亡率的食源性疫情。乳链菌肽（Nisin）是一种天然的抗菌物质，被广泛用作食品防腐剂。然而，单增李斯特菌对乳链菌肽产生耐药性的机制尚未完全明确。我们构建了单增李斯特菌的转座子文库，并通过转座子测序（Tn-seq）分析，鉴定出 99 个与天然耐药性相关的基因。为了验证 Tn-seq 结果的准确性，我们在单增李斯特菌中构建了五个突变株（Δ、Δ、Δ、Δ 和 Δ）。结果表明，与脂磷壁酸相关的基因和是抵抗乳链菌肽所必需的，而和突变株对乳链菌肽的耐药性显著增强。在乳链菌肽处理下，Δ 和 Δ 突变株的表面呈现出密集的皱缩、塌陷和膜破裂。和的缺失改变了表面电荷，并降低了对一般应激条件和细胞包膜作用的抗菌药物的耐药性。此外，YqgS 和 LafA 是单增李斯特菌生物膜形成和细胞侵袭所必需的。综上所述，这些结果揭示了单增李斯特菌对乳链菌肽耐药的新机制，并为开发针对食源性耐药病原体的食品级乳链菌肽抑制剂提供了独特的靶标。单增李斯特菌是一种机会性革兰氏阳性病原体，可引起李斯特菌病，广泛存在于各种食品中，包括即食食品、肉类和乳制品。乳链菌肽是唯一一种经 FDA 批准可作为食品级抑制剂在 50 多个国家使用的类细菌素。先前的研究表明，与其他革兰氏阳性病原体相比，单增李斯特菌对乳链菌肽介导的杀菌作用更具耐药性。然而，单增李斯特菌对乳链菌肽产生耐药性的机制尚未完全明确。在这里，我们使用 mariner 转座子文库通过转座子测序在全基因组范围内鉴定与乳链菌肽耐药相关的基因。我们首次发现 YqgS 和 LafA（脂磷壁酸相关蛋白）是抵抗乳链菌肽所必需的。随后，我们研究了 YqgS 和 LafA 在单增李斯特菌应激抵抗、抗菌药物耐药性、生物膜形成和在哺乳动物细胞中的毒力方面的作用。
