鼠伤寒沙门菌T3SS效应蛋白AvrA通过抑制衣康酸递送增强其胞内存活

doi:10.3969/j.issn.1673-8640.2026.06.009

摘要/Abstract

摘要：

目的筛选并鉴定影响宿主细胞抗菌代谢物衣康酸转运的鼠伤寒沙门菌Ⅲ型分泌系统（T3SS）效应蛋白。方法构建稳定表达衣康酸合成关键酶免疫应答基因1蛋白（IRG1）的HeLa细胞系，转染表达沙门菌效应蛋白后进行细菌感染，通过平板菌落计数和衣康酸荧光素酶报告系统检测衣康酸向含沙门菌囊泡（SCV）内的递送水平。采用自杀质粒同源重组法构建效应蛋白基因敲除的沙门菌突变株，结合平板菌落计数和衣康酸荧光素酶报告实验、共聚焦成像结果，进一步分析衣康酸递送变化。采用实时荧光定量聚合酶链反应（PCR）和蛋白质免疫印迹法检测效应蛋白AvrA对IRG1在mRNA和蛋白水平表达的影响。通过共聚焦成像明确AvrA在细胞内的定位。采用液相色谱-串联质谱（LC-MS/MS）技术筛选AvrA潜在的宿主互作蛋白。结果效应蛋白AvrA显著抑制衣康酸向SCV的递送（P<0.01）。在HeLa-IRG1细胞中过表达AvrA后，衣康酸递送效率下降（P<0.001）；感染AvrA敲除菌株的细胞中，衣康酸递送水平显著升高（P<0.001），且细菌在巨噬细胞内的存活能力下降（P<0.001）。实时荧光定量PCR和蛋白质免疫印迹法结果显示，AvrA不影响IRG1的表达。亚细胞定位分析结果显示，AvrA同时定位于宿主细胞质和细胞核。LC-MS/MS初步筛选出多个可能的AvrA宿主互作蛋白。结论鼠伤寒沙门菌通过其T3SS分泌的效应蛋白AvrA抑制衣康酸向SCV内的递送，从而逃逸宿主细胞自主免疫应答，增强细菌在宿主细胞内的生存能力。该过程不依赖于对IRG1表达的调控。

关键词: 沙门菌, Ⅲ型分泌系统, 效应蛋白, AvrA, 细胞自主免疫应答, 衣康酸

Abstract:

Objective To screen and identify type Ⅲ secretion system（T3SS） effector protein of Salmonella typhimurium that affect the transfection of host cell itaconate. Methods A stable HeLa cell line expressing the key enzyme immune responsive gene 1（IRG1） for itaconate synthesis was constructed. After transfection with the effector protein of Salmonella，bacterial infection was performed. The transfection level of itaconate into Salmonella-containing vesicles（SCV） was determined by plate colony counting and the luciferase reporter system for itaconate. A mutant strain of Salmonella with effector protein gene knockout was constructed by homologous recombination using suicide plasmids. Combined with colony counting，luciferase reporter experiments and confocal imaging，the changes in itaconate transfection were further analyzed. Real-time fluorescence quantitative polymerase chain reaction（qPCR） and western blotting were used to determine the effect of effector protein AvrA on the expression of IRG1 at the mRNA and protein levels. Confocal imaging was used to determine the localization of AvrA in the cells. Liquid chromatography-tandem mass spectrometry（LC-MS/MS） was used to screen potential host-interacting proteins of AvrA. Results The effector protein AvrA significantly inhibited itaconate transfection into SCV（P<0.01）. After overexpression of AvrA in HeLa-IRG1 cells，the transfection efficiency of itaconate decreased（P<0.001）. In cells infected with the AvrA knockout strain，the transfection level increased（P<0.001），and the survival ability of bacteria in macrophages decreased（P<0.001）. The real-time fluorescence quantitative PCR and western blotting showed that AvrA did not affect the expression of IRG1. Subcellular localization analysis showed that AvrA was simultaneously located in the host cytoplasm and nucleus. LC-MS/MS initially screened multiple potential host-interacting proteins of AvrA. Conclusions Salmonella typhimurium secretes the effector protein AvrA through its T3SS to inhibit the itaconate transfection into the SCV，thereby escaping the host cell's autonomous immune response and enhancing the survival ability of bacteria in the host cell. This process does not rely on the regulation of IRG1 expression.

Key words: Salmonella, Type Ⅲ secretion system, Effector protein, AvrA, Host autonomous immune response, Itaconate

中图分类号:

R378.2

赵翃宇, 钟梦娇, 李一荣, 连欢. 鼠伤寒沙门菌T3SS效应蛋白AvrA通过抑制衣康酸递送增强其胞内存活[J]. 检验医学, 2026, 41(6): 575-582.

ZHAO Hongyu, ZHONG Mengjiao, LI Yirong, LIAN Huan. Salmonella typhimurium T3SS effector protein AvrA enhances its intracellular survival by inhibiting itaconate transfection[J]. Laboratory Medicine, 2026, 41(6): 575-582.

图/表 8

表1 引物序列

基因名称	前向引物（5'→3'）	反向引物（5'→3'）
avrA-US	AACTGCAGCCCGGGGGATCCGAACGCCCTT- ATGGTTTC	CTGAAAAGTTAAAGTGAAATTATAAGG- TTGCCTGC
avrA-DS	ACCTTATAATTTCACTTTAACTTTTCAGATTT- AATATATTGCC	CCCACCGCGGTGGCGGCCGCCGGTAT- TAACTGGACTAATTGTT

表2 引物序列

基因名称	前向引物（5'→3'）	反向引物（5'→3'）
Irg1	CTGCCACTCAGACTAAGCCC	GATGTGCCGGGAAGCTCTTA
Gapdh	ACGGCCGCATCTTCTTGTGCA	ACGGCCAAATCCGTTCACACC

图1 沙门菌T3SS效应蛋白的筛选注：（a） HeLa-IRG1细胞中IRG1蛋白表达的免疫印迹图；（b）基于stm3121蛋白的衣康酸生物传感器示意图；（c）沙门菌T3SS效应蛋白筛选流程示意图；（d）不同效应蛋白对HeLa-IRG1细胞中衣康酸递送影响的筛选结果。

图2 AvrA在HeLa-IRG1细胞中对衣康酸递送的影响注：（a） SL1344-AvrA和LT2-AvrA对HeLa-IRG1细胞中衣康酸递送至SCV的影响；（b）HeLa-IRG1细胞中IRG1和AvrA表达的免疫印迹图。

图3 鼠伤寒沙门菌LT2-ΔavrA菌株的构建与鉴定注：（a）携带纳米荧光素酶报告基因的鼠伤寒沙门菌SL1344和LT2菌株中生物传感器的响应情况；鼠伤寒沙门菌SL1344+空载；鼠伤寒沙门菌SL1344+衣康酸感受器；鼠伤寒沙门菌LT2+空载；鼠伤寒沙门菌LT2+衣康酸感受器；（b）携带eGFP报告基因的鼠伤寒沙门菌SL1344和LT2菌株中生物传感器的响应情况；鼠伤寒沙门菌SL1344+空载；鼠伤寒沙门菌SL1344+衣康酸感受器；鼠伤寒沙门菌LT2+空载；鼠伤寒沙门菌LT2+衣康酸感受器；（c）LT2-ΔavrA敲除菌株的PCR鉴定策略示意图；（d）电泳结果；泳道1~7为候选敲除菌株；泳道G为野生型鼠伤寒沙门菌LT2基因组DNA对照。

图4 AvrA敲除促进衣康酸向SCV递送并降低细菌在巨噬细胞内的存活注：（a）纳米荧光素酶检测敲除AvrA对巨噬细胞中衣康酸向SCV递送的影响；（b）敲除AvrA对鼠伤寒沙门氏菌LT2在巨噬细胞内存活的影响；（c）共聚焦成像检测敲除AvrA对巨噬细胞中衣康酸向SCV递送的影响；比例尺5 μm。

图5 沙门菌效应蛋白AvrA对IRG1表达无影响注：（a）LT2-野生型、LT2-ΔavrA感染RAW264.7内Irg1 mRNA相对表达量比较；●LT2-野生型；●LT2-ΔavrA；（b）LT2-野生型、LT2-ΔavrA感染RAW264.7内IRG1和β-actin蛋白表达的免疫印迹图。

图6 质谱筛选鉴定AvrA的宿主互作蛋白注：（a）AvrA表达后宿主差异蛋白富集情况的火山图；（b）AvrA在HeLa细胞内的定位情况；比例尺5 μm。

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