Table_2_Iron Limitation in Klebsiella pneumoniae Defines New Roles for Lon Protease in Homeostasis and Degradation by Quantitative Proteomics.XLSX

Nutrient adaptation is key in limiting environments for the promotion of microbial growth and survival. In microbial systems, iron is an essential component for many cellular processes, and bioavailability varies greatly among different conditions. In the bacterium, Klebsiella pneumoniae, the impact of iron limitation is known to alter transcriptional expression of iron-acquisition pathways and influence secretion of iron-binding siderophores, however, a comprehensive view of iron limitation at the protein level remains to be defined. Here, we apply a mass-spectrometry-based quantitative proteomics strategy to profile the global impact of iron limitation on the cellular proteome and extracellular environment (secretome) of K. pneumoniae. Our data define the impact of iron on proteins involved in transcriptional regulation and emphasize the modulation of a vast array of proteins associated with iron acquisition, transport, and binding. We also identify proteins in the extracellular environment associated with conventional and non-conventional modes of secretion, as well as vesicle release. In particular, we demonstrate a new role for Lon protease in promoting iron homeostasis outside of the cell. Characterization of a Lon protease mutant in K. pneumoniae validates roles in bacterial growth, cell division, and virulence, and uncovers novel degradation candidates of Lon protease associated with improved iron utilization strategies in the absence of the enzyme. Overall, we provide evidence of unique connections between Lon and iron in a bacterial system and suggest a new role for Lon protease in the extracellular environment during nutrient limitation.

营养适配是限制环境中促进微生物生长与存活的核心要素。在微生物系统中，铁是诸多细胞过程的必需组分，且其生物可利用性在不同环境条件下差异悬殊。对于肺炎克雷伯菌（Klebsiella pneumoniae），已有研究表明铁限制会改变其铁摄取通路的转录表达，并影响铁结合性铁载体（siderophores）的分泌，但目前学界对蛋白质层面的铁限制调控机制仍有待全面阐明。本研究采用基于质谱的定量蛋白质组学（mass-spectrometry-based quantitative proteomics）策略，系统解析铁限制对肺炎克雷伯菌细胞蛋白质组（proteome）与胞外环境（分泌组，secretome）的全局影响。本研究数据明确了铁对转录调控相关蛋白的调控作用，并着重揭示了铁对大量参与铁摄取、转运及结合过程的蛋白的调控功能。我们还在胞外环境中鉴定到与经典、非经典分泌模式及囊泡释放相关的蛋白。尤为关键的是，本研究证实了Lon蛋白酶（Lon protease）在细胞外促进铁稳态（iron homeostasis）的全新功能。对肺炎克雷伯菌Lon蛋白酶突变体的表征验证了该酶在细菌生长、细胞分裂与毒力（virulence）中的功能，并鉴定出在缺失该酶时可优化细菌铁利用策略的新型Lon蛋白酶降解靶标。综上，本研究证实了细菌系统中Lon蛋白酶与铁之间存在独特关联，并提出了营养限制条件下Lon蛋白酶在胞外环境中的全新作用。