Fagerquist Clifton K, Zaragoza William J
Produce Safety & Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA, United States.
Front Nutr. 2018 Dec 10;5:124. doi: 10.3389/fnut.2018.00124. eCollection 2018.
We performed proteolytic surface-shaving with trypsin on three strains/sevovars of (SEE): Newport, Kentucky, and Thompson. Surfaced-exposed proteins of live bacterial cells were digested for 15 min. A separate 20 h re-digestion was also performed on the supernatant of each shaving experiment to more completely digest protein fragments into detectable peptides for proteomic analysis by nano-liquid chromatography-electrospray ionization-Orbitrap mass spectrometry. Control samples (i.e., no trypsin during surface-shaving step) were also performed in parallel. We detected peptides of flagella proteins: FliC (filament), FliD (cap), and FlgL (hook-filament junction) as well as peptides of FlgM (anti-σ factor), i.e., the negative regulator of flagella synthesis. For SEE Newport and Thompson, we detected pathogenicity island 1 (SPI-1) secreted effector/invasion proteins: SipA, SipB, SipC, and SipD, whereas no Sip proteins were detected in control samples. No Sip proteins were detected for SEE Kentucky (or its control) although genes were confirmed to be present. Our results may suggest a biological response (<15 min) to proteolysis of live cells for these SEE strains and, in the case of Newport and Thompson, a possible invasion response.
我们用胰蛋白酶对肠炎沙门氏菌（SEE）的三个菌株/血清型进行了蛋白水解表面刮削处理：纽波特、肯塔基和汤普森。对活细菌细胞的表面暴露蛋白进行了15分钟的消化。还对每个刮削实验的上清液进行了单独20小时的再消化，以便将蛋白质片段更完全地消化成可检测的肽段，用于通过纳升液相色谱-电喷雾电离-轨道阱质谱进行蛋白质组学分析。同时也平行进行了对照样品（即表面刮削步骤中不添加胰蛋白酶）的处理。我们检测到了鞭毛蛋白的肽段：FliC（鞭毛丝）、FliD（帽）和FlgL（钩-鞭毛连接）以及FlgM（抗σ因子）的肽段，即鞭毛合成的负调节因子。对于SEE纽波特和汤普森菌株，我们检测到了毒力岛1（SPI-1）分泌的效应蛋白/侵袭蛋白：SipA、SipB、SipC和SipD，而在对照样品中未检测到Sip蛋白。对于SEE肯塔基菌株（及其对照），尽管确认存在相关基因，但未检测到Sip蛋白。我们的结果可能表明这些SEE菌株对活细胞蛋白水解有生物学反应（<15分钟），对于纽波特和汤普森菌株而言，可能存在侵袭反应。
