Palace Samantha G, Proulx Megan K, Lu Shan, Baker Richard E, Goguen Jon D
Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
Laboratory of Nucleic Acid Vaccines, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
mBio. 2014 Aug 19;5(4):e01385-14. doi: 10.1128/mBio.01385-14.
Rapid growth in deep tissue is essential to the high virulence of Yersinia pestis, causative agent of plague. To better understand the mechanisms underlying this unusual ability, we used transposon mutagenesis and high-throughput sequencing (Tn-seq) to systematically probe the Y. pestis genome for elements contributing to fitness during infection. More than a million independent insertion mutants representing nearly 200,000 unique genotypes were generated in fully virulent Y. pestis. Each mutant in the library was assayed for its ability to proliferate in vitro on rich medium and in mice following intravenous injection. Virtually all genes previously established to contribute to virulence following intravenous infection showed significant fitness defects, with the exception of genes for yersiniabactin biosynthesis, which were masked by strong intercellular complementation effects. We also identified more than 30 genes with roles in nutrient acquisition and metabolism as experiencing strong selection during infection. Many of these genes had not previously been implicated in Y. pestis virulence. We further examined the fitness defects of strains carrying mutations in two such genes-encoding a branched-chain amino acid importer (brnQ) and a glucose importer (ptsG)-both in vivo and in a novel defined synthetic growth medium with nutrient concentrations matching those in serum. Our findings suggest that diverse nutrient limitations in deep tissue play a more important role in controlling bacterial infection than has heretofore been appreciated. Because much is known about Y. pestis pathogenesis, this study also serves as a test case that assesses the ability of Tn-seq to detect virulence genes.
Our understanding of the functions required by bacteria to grow in deep tissues is limited, in part because most growth studies of pathogenic bacteria are conducted on laboratory media that do not reflect conditions prevailing in infected animal tissues. Improving our knowledge of this aspect of bacterial biology is important as a potential pathway to the development of novel therapeutics. Yersinia pestis, the plague bacterium, is highly virulent due to its rapid dissemination and growth in deep tissues, making it a good model for discovering bacterial adaptations that promote rapid growth during infection. Using Tn-seq, a genome-wide fitness profiling technique, we identified several functions required for fitness of Y. pestis in vivo that were not previously known to be important. Most of these functions are needed to acquire or synthesize nutrients. Interference with these critical nutrient acquisition pathways may be an effective strategy for designing novel antibiotics and vaccines.
深层组织中的快速生长对于鼠疫耶尔森菌（鼠疫的病原体）的高毒力至关重要。为了更好地理解这种异常能力背后的机制，我们使用转座子诱变和高通量测序（Tn-seq）来系统地探究鼠疫耶尔森菌基因组中对感染期间适应性有贡献的元件。在完全有毒力的鼠疫耶尔森菌中产生了超过一百万个代表近200,000种独特基因型的独立插入突变体。对文库中的每个突变体进行体外在丰富培养基上以及静脉注射后在小鼠体内增殖能力的检测。实际上，先前确定的在静脉感染后对毒力有贡献的所有基因都显示出明显的适应性缺陷，但耶尔森菌素生物合成基因除外，其被强烈的细胞间互补效应所掩盖。我们还鉴定出30多个在营养获取和代谢中起作用的基因在感染期间经历了强烈选择。这些基因中的许多以前未被认为与鼠疫耶尔森菌的毒力有关。我们进一步研究了携带编码支链氨基酸转运蛋白（brnQ）和葡萄糖转运蛋白（ptsG）的两个此类基因突变的菌株在体内以及在一种新型的定义合成生长培养基中的适应性缺陷，该培养基中的营养浓度与血清中的浓度相匹配。我们的研究结果表明深层组织中多种营养限制在控制细菌感染中所起的作用比以往认识到的更为重要。由于对鼠疫耶尔森菌的发病机制了解很多，这项研究还作为一个测试案例，评估了Tn-seq检测毒力基因的能力。
我们对细菌在深层组织中生长所需功能的理解有限，部分原因是大多数病原菌的生长研究是在不能反映感染动物组织中普遍存在情况的实验室培养基上进行的。作为开发新型疗法的潜在途径之一，提高我们对细菌生物学这一方面的认识很重要。鼠疫耶尔森菌作为鼠疫杆菌，由于其在深层组织中的快速传播和生长而具有高毒力，使其成为发现促进感染期间快速生长的细菌适应性的良好模型。使用全基因组适应性分析技术Tn-seq，我们鉴定出鼠疫耶尔森菌在体内适应性所需的几种以前未知其重要性的功能。这些功能中的大多数是获取或合成营养所必需的。干扰这些关键的营养获取途径可能是设计新型抗生素和疫苗的有效策略。
