Laboratório de Bioquímica e Microbiologia Aplicada, Instituto de Ciência e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Laboratório de Câncer e Neurobiologia, Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil.
J Proteomics. 2020 Aug 30;226:103906. doi: 10.1016/j.jprot.2020.103906. Epub 2020 Jul 21.
In this work, a comparative analysis of the peripheral cell component (PCC) proteins of Listeria monocytogenes was carried out. The study was conducted on two set of samples consisting of bacteria treated with sub-lethal concentration of nisin and untreated bacteria as control. PCC proteins were extracted by Tris-Urea-EDTA treatment and then subjected to trypsin digestion and mass spectrometry analysis. The whole cell proteome was analyzed through label-free quantitative proteomics approach. Proteomic analysis was carried out using OrbiTrap Mass Spectrometer coupled to nanoflow liquid chromatography. The treatment with sub-lethal nisin concentration resulted in 62 up regulated and 97 down regulated proteins compared to untreated samples. Using PSORTb 3.0, 19 and 18 surface proteins were detected among the up regulated and down regulated proteins, respectively. Proteins related with increased biofilm formation by L.monocytogenes, such as moonlight proteins of the pyruvate dehydrogenase complex and flagellin-related proteins, were identified as up regulated surface proteins. Proteins associated with virulence of L.monocytogenes, including listeriolysin O, internalin B and actin assembly-inducing protein, were detected among the down regulated proteins. To confirm proteomics data, increased production of biofilm was experimentally confirmed in nisin-treated cells through crystal violet method. BIOLOGICAL SIGNIFICANCE: Proteosurfaceomics can be defined as the "omics" science applied to the proteins of the peripheral cell component (PCC). The surface proteins of Listeria monocytogenes, an important foodborne pathogen were investigated after treatment with nisin, a bacteriocin approved as a natural food preservative by regulatory agencies. Recent cases of nisin tolerance by Listeria spp. were documented, and deeper studies on the molecular process behind the bacterial survival may help in both understanding the development of tolerance process and comparing nisin effect with other antimicrobial compounds.
本工作对李斯特菌的外周细胞成分（PCC）蛋白进行了比较分析。该研究基于两组样本进行，一组为经亚致死浓度乳链菌肽处理的细菌，另一组为未经处理的细菌作为对照。通过 Tris-Urea-EDTA 处理提取 PCC 蛋白，然后进行胰蛋白酶消化和质谱分析。采用无标记定量蛋白质组学方法分析全细胞蛋白质组。使用 OrbiTrap 质谱仪与纳流液相色谱联用进行蛋白质组分析。与未经处理的样品相比，用亚致死浓度乳链菌肽处理后，有 62 个蛋白上调，97 个蛋白下调。使用 PSORTb 3.0，分别在上调和下调的蛋白中检测到 19 个和 18 个表面蛋白。鉴定到与李斯特菌生物膜形成增加相关的蛋白，如丙酮酸脱氢酶复合物的月光蛋白和鞭毛相关蛋白，这些蛋白被鉴定为上调的表面蛋白。与李斯特菌毒力相关的蛋白，包括溶血素 O、内化素 B 和肌动蛋白组装诱导蛋白，被鉴定为下调蛋白。为了验证蛋白质组学数据，通过结晶紫法实验证实了经乳链菌肽处理的细胞中生物膜的产量增加。生物学意义：蛋白质组表面组学可定义为应用于外周细胞成分（PCC）蛋白的“组学”科学。研究了食品中重要病原体李斯特菌在乳链菌肽处理后的表面蛋白，乳链菌肽是一种经监管机构批准的天然食品防腐剂。最近有报道称李斯特菌对乳链菌肽产生了耐受性，深入研究细菌存活背后的分子过程有助于了解耐受过程的发展，并将乳链菌肽的作用与其他抗菌化合物进行比较。
