New insights into the stress response mechanisms of stress-resistant Listeria monocytogenes via multi-omics and cell morphological changes

Listeria monocytogenes (Lm) can persist in stressful conditions such as low pH, temperature, and salt concentration. Omics approaches enable the investigation of biomolecular features at both the transcript and protein levels. This study aimed to understand stress response mechanisms by analyzing transcript and protein levels of stress-resistant L. monocytogenes (SR-Lm) under multiple stress conditions. RNA sequencing was performed at 4, 8, 12, 24, and 48 h growth under a multiple stress condition (pH 3 + 1 °C + 5% salt) and RT-qPCR was also conducted. Gene expression analysis revealed upregulation of the yebS, tsx, secA, gap, gtfA, along with several stress-related genes (cspB, cspD, spxA, and cysK) during 48 h. Protein expression profiles of SR-Lm were conducted under (1) 5% salt+1 °C, (2) pH 3 + 1 °C, and (3) pH 3 + 1 °C + 5% salt conditions at 4 and 12 h. Some proteins including Gap, Eno, Citz, and PurM were upregulated in both analyses, potentially associated with stress-related biomarkers. KEGG pathway analysis was performed using proteomic data to predict stress response mechanisms including chaperone, glycolysis, and glutamate decarboxylase system (GAD). Cell morphology under stress and normal conditions was observed, with some elongated, thin, and bumpy cell shapes under acid and multiple stress conditions. This study provides new insights into the stress response of SR-Lm, enhancing the understanding of how Lm survives in adverse food processing and storage environments.单核细胞增生李斯特菌（Listeria monocytogenes, Lm）可在低pH、温度胁迫及盐浓度胁迫等严苛环境中持续存活。组学方法（Omics approaches）能够实现在转录组与蛋白质组层面解析生物分子特征。本研究旨在通过分析多重胁迫条件下抗胁迫单核细胞增生李斯特菌（Stress-resistant L. monocytogenes, SR-Lm）的转录与蛋白质表达水平，解析其胁迫应答机制。本研究在多重胁迫条件（pH 3 + 1 ℃ + 5%氯化钠）下，于培养4、8、12、24及48小时时进行RNA测序（RNA sequencing），同时开展了实时荧光定量PCR（RT-qPCR）实验。基因表达分析结果显示，培养48小时时，yebS、tsx、secA、gap、gtfA等基因以及cspB、cspD、spxA、cysK等多个胁迫相关基因均出现上调表达。本研究对SR-Lm在三种胁迫条件下的蛋白质表达谱进行了分析，检测时点为培养4小时与12小时：(1) 5%氯化钠+1 ℃；(2) pH 3 + 1 ℃；(3) pH 3 + 1 ℃ + 5%氯化钠。两种组学分析均检测到Gap、Eno、Citz及PurM等蛋白质出现上调表达，这些蛋白可能作为胁迫相关生物标志物（biomarkers）。本研究利用蛋白质组数据开展京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路分析，预测了包括分子伴侣系统、糖酵解通路及谷氨酸脱羧酶系统（Glutamate decarboxylase system, GAD）在内的胁迫应答机制。研究同时观察了胁迫与正常条件下的细胞形态：在酸性及多重胁迫条件下，菌体呈现出伸长、纤细且表面凹凸不平的形态。本研究为解析SR-Lm的胁迫应答机制提供了新视角，有助于加深对Lm在不良食品加工与储存环境中存活机制的理解。