铜绿假单胞菌Ⅵ型分泌系统效应因子 TseL 的功能鉴定

铜绿假单胞菌是一种常见的条件致病菌，经常引起住院病人的院内感染，同时也是COVID-19患者中常见的并发感染病原体。Ⅵ型分泌系统(T6SS)是铜绿假单胞菌重要的毒力机制，它负责向竞争微生物或宿主细胞注入多种毒力效应因子。前期，在COVID-19患者痰液样本中分离得到一株高毒力的铜绿假单胞菌临床菌株LYSZa7。通过对LYSZa7基因组注释分析，本研究发现了一个潜在的T6SS的脂酶效应因子基因簇，并且该基因簇广泛存在于铜绿假单胞菌中，但在实验室模型株PAO1/PA14中不存在。本项目确认了该效应因子的脂酶功能和关键结构域，将编码该效应因子的基因命名为tseL。随后，对TseL的功能分析发现，TseL靶向细菌周质引起膜损伤效应，参与铜绿假单胞菌的种内和种间竞争，同时能够靶向宿主细胞。另外，铜绿假单胞菌LYSZa7还编码两个可以中和其毒性的免疫蛋白基因（tsiP1，tsiP2）。这两个免疫蛋白通过生物化学结合均能保护细菌自身免受TseL的伤害。综上所述，本项目发现了在铜绿假单胞菌临床菌株致病性和竞争中起到作用的T6SS效应蛋白TseL及其免疫蛋白，并提出TseL在种内/间竞争发挥一定作用并且能够增加对细胞的感染性。该研究成果为铜绿假单胞菌临床菌株的流行病学鉴定提供了新的分子标记。

Pseudomonas aeruginosa is a predominant opportunistic pathogen that often causes nosocomial infections in hospitalized patients. It is identified as a common coinfecting pathogen in COVID-19 patients causing exacerbation of illness. The type VI secretion system (T6SS) is an important virulence mechanism of P. aeruginosa, and it is responsible for injection of numerous virulence effectors into competing microbes or host cells. This work identified a T6SS effector, TseL, from a clinical P. aeruginosa strain LYSZa7, which is isolated from sputum samples of a COVID-19 patient. Also, this effector was found ubiquitous in P. aeruginosa but not in the lab model strain PAO1/PA14. The TseL contains a lipase domain and was proved to have T6SS-dependent lipase activity. The TseL contributed to intra- and inter-species competition by targeting bacterial periplasm to cause membrane damages. TseL also contributed to P. aeruginosa LYSZa7 virulence against eukaryotic cells. Moreover, P. aeruginosa LYSZa7 genome also encodes two immunity protein genes (tsiP1, tsiP2) in the same gene cluster, which were shown to neutralize the TseL toxicity to protect the host bacterial cells. Taken together, our findings showed that the novel P. aeruginosa T6SS-delivered phospholipase effector TseL is active against both prokaryotic and eukaryotic cells, highlighting the diversity of effectors of the T6SS systems in the Pseudomonas aeruginosa clinical strains. This study provides a new molecular marker for the epidemiological identification of clinical strains of P. aeruginosa.

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