T3SS通过ERK/ROS信号通路促进铜绿假单胞菌侵袭肺上皮细胞的作用

熊浚智; 余华; 王兴敏; 何晓梅; 代黔; 邱静

doi:10.16016/j.2097-0927.202407056

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T3SS通过ERK/ROS信号通路促进铜绿假单胞菌侵袭肺上皮细胞的作用

基础医学 | 更新时间：2026-01-09

- T3SS通过ERK/ROS信号通路促进铜绿假单胞菌侵袭肺上皮细胞的作用
- Role of T3SS in promoting Pseudomonas aeruginosa internalization in pulmonary epithelial cells via ERK/ROS signaling pathway
- 陆军军医大学学报 2024年46卷第22期页码：2493-2504
- 作者机构：
  
  1.400037 重庆，陆军军医大学(第三军医大学)第二附属医院：临床医学研究中心
  2.400037 重庆，陆军军医大学(第三军医大学)第二附属医院：药剂科
- 作者简介：
  
  [邱静，E-mail: 375895062@tmmu.edu.cnQIU Jing, E-mail: 375895062@tmmu.edu.cn
- 基金信息：
  
  重庆市自然科学基金面上项目(CSTB2023NSCQ-MSX0931)
- DOI：10.16016/j.2097-0927.202407056
  中图分类号： R363.21;R378.991;R563.9
- 收稿：2024-09-26，
  
  修回：2024-10-20，
  
  纸质出版：2024-11-30
- 稿件说明：
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熊浚智, 余华, 王兴敏, 等. T3SS通过ERK/ROS信号通路促进铜绿假单胞菌侵袭肺上皮细胞的作用[J]. 陆军军医大学学报, 2024,46(22):2493-2504.

Junzhi XIONG, Hua YU, Xingmin WANG, et al. Role of T3SS in promoting Pseudomonas aeruginosa internalization in pulmonary epithelial cells via ERK/ROS signaling pathway[J]. Journal of Army Medical University, 2024, 46(22): 2493-2504.
熊浚智, 余华, 王兴敏, 等. T3SS通过ERK/ROS信号通路促进铜绿假单胞菌侵袭肺上皮细胞的作用[J]. 陆军军医大学学报, 2024,46(22):2493-2504. DOI： 10.16016/j.2097-0927.202407056.

Junzhi XIONG, Hua YU, Xingmin WANG, et al. Role of T3SS in promoting Pseudomonas aeruginosa internalization in pulmonary epithelial cells via ERK/ROS signaling pathway[J]. Journal of Army Medical University, 2024, 46(22): 2493-2504. DOI： 10.16016/j.2097-0927.202407056.

摘要

目的

探究Ⅲ型分泌型系统(type three secretion system，T3SS)在调控铜绿假单胞菌(

Pseudomonas aeruginosa

，PA)侵袭肺上皮细胞中的作用及具体机制。

方法

采用PA菌株感染人非小细胞肺癌A549细胞，分为未处理组、PAO1(PA实验室标准菌株)组、△

exsA

(缺失与T3SS转录激活相关的基因

exsA

)组、△

pscJ

(缺失与T3SS蛋白分泌相关基因

pscJ

)组和PAO1-E(经EGTA诱导后高表达T3SS基因)感染组。采用U0126抑制A549细胞ERK活性或通

过apocynin(APO)/N-acetyl-L-cysteine(NAC)抑制A549细胞活性氧(reactive oxygen species

ROS)产生并随后感染PAO1或PAO1-E菌株，分为未处理组、PAO1或PAO1-E感染组、抑制剂处理组，以及抑制剂联合PAO1或PAO1-E感染组。选用庆大霉素杀伤胞外细菌并将细胞裂解液倍比稀释后涂布PA筛选平板，通过计数菌落形成单位(CFUs)，明确PA感染后细胞内的细菌量；通过荧光探针标记以及流式细胞术检测感染后细胞内ROS水平；通过Western blot检测感染后ERK通路的活化情况。

结果

与PAO1感染组相比，△

exsA

和△

pscJ

感染组的细胞内细菌量减少(

＜0.05，

＜0.01)，且伴随ROS产生减少(

＜0.01)，而PAO1-E感染组则呈现相反趋势(

＜0.01)。PAO1或PAO1-E感染联合APO/NAC处理组的细胞内细菌量明显低于PAO1或PAO1-E感染组(

＜0.01)。相较PAO1感染组，△

exsA

和△

pscJ

感染组的ERK磷酸化水平增加(

＜0.01)，而PAO1-E感染组的ERK磷酸化降低(

＜0.01)。与PAO1感染组相比，PAO1联合U0126处理组的细胞ERK磷酸化水平降低，伴随ROS产生以及细胞内细菌量增加(

＜0.01)。

结论

T3SS介导ERK通路抑制可通过促进肺上皮细胞ROS产生，增加PA对肺上皮细胞侵袭。

Abstract

Objective

To explore the role and underlying mechanism of type Ⅲ secretion system (T3SS) in regulating the internalization of

Pseudomonas aeruginosa

(PA) into pulmonary epithelial cells.

Methods

The human non-small cell lung cancer A549 cells were infected with or without PA strains

including wild-type PAO1 (a standard experimental PA strain)

△

exs

A (knockout of the critical activator for T3SS genes)

△

psc

J (T3SS secretion-defective strain) and PAO1-E (EGTA-induced high expression of T3SS genes). The A549 cells pretreated with ERK inhibitor U0126 or reactive oxygen species (ROS) inhibitor apocynin (APO)/N-acetyl-L-cysteine (NAC) were infected with PAO1 or PAO1-E strain. Thus

the experiment was grouped as follows: the mock-treated group

PAO1- or PAO1-E-infected group

inhibitor-treated group

and PAO1/PAO1-E plus inhibitor-treated group. Extracellular bacteria were killed by gentamicin

and the cell lysates were diluted and then plated on PA screening pla

tes. Bacterial amounts were detected by counting colony-forming units (CFUs). The production of ROS was analyzed using fluorescent probe labeling and flow cytometry. The activation of the ERK pathway was detected by Western blotting.

Results

Compared with the PAO1-infected group

the intracellular bacteria and ROS level in △

exs

A- or △

psc

J-infected cells were lower (

0.05

0.01)

so was the generation of ROS(

0.01); In contrast

those of the PAO1-E strain-infected cells displayed an opposite trend (

0.01). Compared with the PAO1- or PAO1-E-infected group

the cells pretreated with APO/NAC followed by PAO1 or PAO1-E infection showed reduced intracellular bacterial amounts (

0.01). Compared to the PAO1-infected A549 cells

the phosphorylation level of ERK was increased in the △

exs

A- or △

psc

J-infected cells (

0.01)

while that level was suppressed in the PAO1-E-treated cells (

0.01). Compared with the PAO1-infected group

the PAO1-infected cells pretreated with U0126 displayed reduced ERK activation

elevated ROS production

and increased intracellular counts of PAO1 (

0.01).

Conclusion

T3SS-mediated inhibition of the ERK pathway promotes the production of ROS and the internalization of PA in lung epithelial cells.

关键词

Keywords

references

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