LisK/R通过调控鞭毛基因表达影响单增李斯特菌低温生长

LisK/R plays a role in Listeria monocytogenes growth at low temperatures by regulating the expression of flagellar genes

摘要:

目的 单增李斯特菌(Listeria monocytogenes, Lm)是一种革兰阳性食源性病原菌，可导致病死率高达20%-30%的人兽共患病——李斯特菌病(listeriosis)。Lm能够在低温下生长繁殖，对食品安全和人类健康构成极大的威胁。探究Lm低温生长机制，将为制定控制Lm低温生长的措施以及研发低温生长抑制剂提供理论依据。研究发现细菌双组分系统(two-component signal transduction system, TCS) LisK/R对Lm低温生长具有调控作用，但其具体机制尚未明确。因此，本研究旨在探讨LisK/R对Lm低温生长的作用机制。 方法 以Lm食品分离株LM201为亲本株，构建3个菌株：ΔlisR (LisK/R的RR缺失株)、ΔlisRc (ΔlisR的回补株)和ΔlisKc [LisK/R的HK缺失株ΔlisK (课题组之前已构建)的回补株]。测定亲本株、缺失株和回补株在4 ℃下的生长曲线；对4 ℃下生长的缺失株ΔlisK和亲本株进行RNA-Seq及数据分析；在4 ℃下，用软琼脂平板测定亲本株、缺失株和回补株的菌株运动圈直径，用透射电镜观察菌体鞭毛形成情况，用RT-qPCR测定鞭毛基因表达量。 结果 在4 ℃低温条件下，缺失株的生长速率显著低于亲本株(P<0.05)，而回补株的生长情况与亲本株一致。RNA-Seq基因表达差异性分析结果显示，与亲本株相比，缺失株ΔlisK的鞭毛基因表达普遍显著上调(P<0.05)。在4 ℃低温条件下，缺失株的运动圈直径显著大于亲本株和回补株(P<0.05)；电镜观察的结果显示，缺失株的鞭毛数量多，而亲本株和回补株的鞭毛数量较少；RT-qPCR测定结果也显示，缺失株的鞭毛基因表达量显著高于亲本株和回补株(P<0.05)。 结论 在4 ℃低温条件下，生长速率低的LisK/R缺失株鞭毛数量多，而生长速率高的亲本株鞭毛数量少，表明Lm通过LisK/R抑制鞭毛基因的表达，减少鞭毛产量，从而将能量用于生长以促进Lm在低温下的生长，其具体的分子调控机制尚需进一步研究。本研究为阐明Lm低温生长机制提供了新的信息。

关键词:单增李斯特菌;低温生长;双组分系统LisK/R;鞭毛

Abstract:

Objective Listeria monocytogenes (Lm) is a Gram-positive foodborne pathogen causing human and animal listeriosis with high case fatality rates (20%-30%). Lm can grow at low temperatures, which brings great risks to food safety and human health. Exploring the mechanism of Lm growth at low temperatures will provide a theoretical basis for the formulation of measures to control Lm growth at low temperatures and the development of low-temperature growth inhibitors. Studies have shown that LisK/R, a two-component signal transduction system (TCS) in Lm, plays a role in regulating the growth of Lm at low temperatures, while the specific mechanism remains unclear. This study aims to reveal the mechanism of LisK/R in regulating the growth of Lm at low temperatures. Methods With the Lm strain LM201 isolated from foodstuff as the parental strain, we constructed three strains: ΔlisR (RR-deleted mutant of LisK/R), ΔlisRc (complementary strain of ΔlisR), and ΔlisKc (complementary strain of ΔlisK, which was constructed before this study in our laboratory). The growth curves of these strains were determined at 4 ℃. RNA-Seq was performed for ΔlisK and the parental strain growing at 4 ℃, and the obtained data were analyzed. We assayed the swarming area diameter on soft agar plate, flagellar biosynthesis by transmission electron microscopy, and transcriptional levels of flagellar genes by RT-qPCR for all the strains at 4 ℃. Results The growth of deletion mutants was slower than that of the parental strain (P<0.05), and the growth of complementary strains was consistent with that of the parental strain at 4 ℃. The RNA-Seq data showed that compared with that in the parental strain, the expression of flagellar genes in ΔlisK was up-regulated (P<0.05). At 4 ℃, the swarming area diameters of deletion mutants on soft agar plates were bigger than those of the parental strain and complementary strains (P<0.05); the deletion mutants had more flagella, while the parental and complementary strains had fewer flagella; the expression of flagellar genes in the deletion mutants was higher than that in the parental and complementary strains (P<0.05). Conclusion At 4 ℃, the LisK/R mutants with slow growth had more flagella, while the parental strain with rapid growth had fewer flagella. The results indicate that LisK/R inhibits the expression of flagellar genes in Lm to reduce flagellar production, and the energy is used to promote Lm growth at low temperatures. The specific molecular regulatory mechanism of LisK/R needs further study. This study provides new information for elucidating the mechanism of Lm growth at low temperatures.

Key words:Listeria monocytogenes;growth at low temperatures;two-component system LisK/R;flagellum