Lawrence Livermore National Laboratory, Livermore, CA, United States.
Sandia National Laboratories, Livermore, CA, United States.
Front Cell Infect Microbiol. 2018 Oct 23;8:370. doi: 10.3389/fcimb.2018.00370. eCollection 2018.
and are the causative agents of melioidosis and glanders, respectively, and are often fatal to humans and animals. Owing to the high fatality rate, potential for spread by aerosolization, and the lack of efficacious therapeutics, and are considered biothreat agents of concern. In this study, we investigate the proteome of , a closely related surrogate for the two more virulent species, during infection of host cells, and compare to that of in culture. Studying the proteome of spp. during infection is expected to reveal molecular mechanisms of intracellular survival and host immune evasion; but proteomic profiling of during host infection is challenging. Proteomic analyses of host-associated bacteria are typically hindered by the overwhelming host protein content recovered from infected cultures. To address this problem, we have applied bio-orthogonal noncanonical amino acid tagging (BONCAT) to , enabling the enrichment of newly expressed bacterial proteins from virtually any growth condition, including host cell infection. In this study, we show that proteins were selectively labeled and efficiently enriched from infected host cells using BONCAT. We also demonstrate that this method can be used to label bacteria by fluorescent tagging. Finally, we present a global proteomic profile of as it infects host cells and a list of proteins that are differentially regulated in infection conditions as compared to bacterial monoculture. Among the identified proteins are quorum sensing regulated genes as well as homologs to previously identified virulence factors. This method provides a powerful tool to study the molecular processes during infection, a much-needed addition to the molecular toolbox.
和 分别是类鼻疽和鼻疽的病原体，对人类和动物通常是致命的。由于高死亡率、气溶胶传播的潜力以及缺乏有效的治疗方法， 和 被认为是值得关注的生物威胁剂。在这项研究中，我们研究了宿主细胞感染期间，与两种更具毒性的 种密切相关的替代物 的蛋白质组，并将其与培养物中的 进行比较。研究 种在感染期间的蛋白质组有望揭示细胞内生存和宿主免疫逃避的分子机制；但是对 种在宿主感染期间的蛋白质组进行研究具有挑战性。从受感染的培养物中回收的宿主蛋白含量极高，这通常会阻碍对宿主相关细菌的蛋白质组分析。为了解决这个问题，我们已经将生物正交非天然氨基酸标记 (BONCAT) 应用于 ，从而可以从几乎任何生长条件（包括宿主细胞感染）中富集新表达的细菌蛋白。在这项研究中，我们表明可以使用 BONCAT 从感染宿主细胞的 中选择性标记和有效富集蛋白质。我们还证明，该方法可用于通过荧光标记来标记 。最后，我们展示了 感染宿主细胞时的全蛋白质组图谱，并列出了与细菌单培养相比在感染条件下差异调节的蛋白质。鉴定出的蛋白质包括群体感应调节基因以及先前鉴定的毒力因子的同源物。该方法为研究 感染期间的分子过程提供了一种强大的工具，是 分子工具包急需的补充。
