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