Gene essentiality profiling reveals a novel determinant of stresses preventing protein aggregation in <i>Salmonella</i>

Adaptation to various stresses during infection is important for <i>Salmonella</i> Typhimurium virulence, while the fitness determinants under infection-relevant stress conditions remain unknown. Here, we simulated conditions <i>Salmonella</i> encountered within the host or in the environment by 15 individual stresses as well as two model cell lines (epithelium and macrophage) to decipher the genes and pathways required for fitness. By high-resolution Tn-seq analysis, a total of 1242 genes were identified as essential for fitness under at least one stress condition. The comparative analysis of fitness determinants in 17 stress conditions indicated the essentiality of genes varied in different mimicking host niches. A total of 12 genes were identified as fitness determinants in all stress conditions, including <i>recB</i>, <i>recC</i>, and <i>xseA</i> (encode three exonuclease subunits necessary for DNA recombination repair) and a novel essential fitness gene <i>yheM</i>. YheM is a putative sulfurtransferase subunit that is responsible for tRNA modification, and our results showed that <i>Salmonella</i> lacking <i>yheM</i> accumulated more aggregates of endogenous protein than wild-type. Moreover, we established a scoring scheme for sRNA essentiality analysis and found STnc2080 of unknown function was essential for resistance to LL-37. In summary, we systematically dissected <i>Salmonella</i> gene essentiality profiling and demonstrated the general and specific adaptive requirements in infection-relevant niches. Our data not only provide valuable insights on how <i>Salmonella</i> responds to environmental stresses during infections but also highlight the potential clinical application of fitness determinants in vaccine development.感染过程中对多种胁迫的适应性对于鼠伤寒沙门氏菌（Salmonella Typhimurium）的毒力至关重要，然而感染相关胁迫条件下的适合度决定因子仍未明确。本研究通过15种单一胁迫以及两种模式细胞系（上皮细胞与巨噬细胞）模拟沙门氏菌在宿主体内或环境中遭遇的条件，以解析其维持适合度所需的基因与通路。通过高分辨率转座子测序（high-resolution Tn-seq）分析，我们共鉴定出1242个在至少一种胁迫条件下对适合度至关重要的基因。对17种胁迫条件下的适合度决定因子进行比较分析后发现，不同模拟宿主微环境中基因的必需性存在差异。共计12个基因被鉴定为所有胁迫条件下的适合度决定因子，包括recB、recC与xseA（分别编码DNA重组修复所需的三种核酸酶亚基）以及一个新的必需适合度基因yheM。YheM是一种假定的硫转移酶亚基，参与tRNA（transfer RNA）修饰；实验结果显示，缺失yheM的沙门氏菌相较于野生型菌株，其内源蛋白聚集物积累量更高。此外，我们建立了一套小RNA（small RNA, sRNA）必需性分析的评分体系，发现功能未知的STnc2080对抵抗LL-37至关重要。综上，我们系统解析了沙门氏菌的基因必需性图谱，并阐明了其在感染相关微环境中的通用与特异性适应性需求。本研究数据不仅为解析沙门氏菌在感染过程中如何响应环境胁迫提供了重要见解，同时也凸显了适合度决定因子在疫苗开发中的潜在临床应用价值。