Adkins Joshua N, Mottaz Heather M, Norbeck Angela D, Gustin Jean K, Rue Joanne, Clauss Therese R W, Purvine Samuel O, Rodland Karin D, Heffron Fred, Smith Richard D
Biological Science Division, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Mol Cell Proteomics. 2006 Aug;5(8):1450-61. doi: 10.1074/mcp.M600139-MCP200. Epub 2006 May 8.
Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes approximately 8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. enterica genome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based "bottom-up" proteomic approach to generate a more complete picture of the gene products that S. typhimurium synthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimurium is known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimurium express genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg(2+) transport proteins than found in other growth conditions. A second more virulent S. typhimurium strain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pdu operon, which encodes enzymes required for propanediol utilization. These pdu operon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdu gene expression contributes to S. typhimurium pathogenesis.
肠炎沙门氏菌鼠伤寒血清型（也称为鼠伤寒沙门氏菌）是一种兼性胞内病原体，在美国每年导致约8000例急性肠胃炎和腹泻的报告病例。尽管已经采用了许多成功的生理学、生物化学和遗传学方法来确定该生物体编码的关键毒力决定因素，但在肠炎沙门氏菌基因组中观察到的大量未表征的阅读框表明，还有更多的毒力因子有待发现。我们使用基于液相色谱-质谱的“自下而上”蛋白质组学方法，以更全面地了解鼠伤寒沙门氏菌在典型实验室条件下以及在已知可诱导毒力基因表达的培养基中合成的基因产物。已知鼠伤寒沙门氏菌在丰富培养基中生长至对数期时会表达许多侵袭上皮细胞所需的基因。相反，鼠伤寒沙门氏菌的稳定期培养物表达全身感染和在受感染巨噬细胞内生长所需的基因。最后，在旨在模拟吞噬泡的酸性、缺镁基本培养基（MgM）中生长的细菌已被证明会上调毒力基因表达。对在每种这些条件下生长的细菌的蛋白质丰度进行的初步比较表明，大多数蛋白质没有显著变化。然而，我们观察到了一些蛋白质亚群，其表达在很大程度上仅限于三种培养条件之一。例如，在MgM中生长的细胞比在其他生长条件下具有更高丰度的Mg(2+)转运蛋白。另一种毒性更强的鼠伤寒沙门氏菌菌株（14028）也在相同的生长条件下培养，并将结果与菌株LT2的结果直接进行比较。这种比较提供了一个独特的机会来对比这些密切相关细菌中的蛋白质群体。在菌株14028中丰度较高的许多蛋白质中，有pdu操纵子的产物，该操纵子编码丙二醇利用所需的酶。这些pdu操纵子蛋白在培养和巨噬细胞感染期间得到了验证。我们的工作为早期观察结果提供了进一步支持，这些观察结果表明pdu基因表达有助于鼠伤寒沙门氏菌发病机制。
