探讨金黄色葡萄球菌（SA）细胞壁锚定蛋白SasX对RNAⅢ转录表达的调控，揭示其对SA的生物膜（BF）形成和致病过程的作用。

收集2022年4~6月南京医科大学附属儿童医院住院患儿临床样本中分离的SA ST239克隆，采用基因敲除和回补技术建立突变株△SasX和回补株△SasX（pRB473-SasX），经RNAⅢ抑制肽（RIP）处理，再采用实时荧光定量PCR（qRT-PCR）检测SasX基因对RNAⅢ转录水平的影响，绘制增殖曲线观察菌株生长速度，镜下观察菌株聚集能力，半定量实验观察BF形成，并采用qPCR验证BF形成相关基因表达。

△SasX株的RNA Ⅲ水平显著低于野生株（t = 4.273、P = 0.037）。△SasX株BF形成能力较野生株显著减弱（t = 4.619、P = 0.032），△SasX（pRB473-SasX）+ RIP株BF形成能力显著低于△SasX（pRB473-SasX）株（t = 7.874、P = 0.011）。△SasX株icaA（t = 5.324、P = 0.027）、sarA（t = 6.250、P = 0.016）和fnbA（t = 4.833、P = 0.031）mRNA水平显著低于野生株；△SasX（pRB473-SasX）+ RIP株icaA（t = 4.386、P = 0.034）和sarA（t = 5.531、P = 0.023）mRNA水平较△SasX（pRB473-SasX）株显著降低。镜下可见野生株有大片聚集成团，△SasX株多为单个散在分布或小范围聚集，△SasX株的聚集能力较野生株显著减弱；△SasX（pRB473-SasX）株的聚集能力和野生株相当，△SasX（pRB473-SasX）+ RIP株的聚集能力较△SasX（pRB473-SasX）株减弱。

SA SasX通过调控RNAⅢ的转录表达，促进细菌的聚集和BF形成能力，从而参与其致病过程。

To investigate the regulation of RNAⅢ transcription by Staphylococcus aureus (SA) surface-anchored protein SasX and its effect on bacterial aggregation and biofilm (BF) formation, which will reveal the effect of SasX on the pathogenicity of SA ST239 clones via RNAⅢ.

SA ST239 HS770 isolated from clinical specimens of hospitalized children of Children’s Hospital of Nanjing Medical University from April to June in 2022 was used to establish mutant strains △SasX and complementary strains △SasX (pRB473-SasX) by gene knockout and complementation technology, and further treated with RNAⅢ inhibitory peptide (RIP). The effect of SasX gene on RNAⅢ transcription level was detected by quantitative real-timePCR (qRT-PCR), and the growth rate of the strains was observed by proliferation curve. The aggregation ability was analyzed by microscopic examination and the BF formation was observed by semi-quantitative BF formation experiments.

The RNAⅢ of △SasX was significantly lower than that of wild strain (t = 4.273, P = 0.037). The BF formation ability of the △SasX was significantly weaker than that of the wild strain (t = 4.619, P = 0.032). The BF-forming ability of the △SasX (pRB473-SasX) + RIP strain was significantly lower than that of the △SasX (pRB473-SasX) (t = 7.874, P = 0.011). The mRNA levels of icaA (t = 5.324, P = 0.027), sarA (t = 6.250, P = 0.016) and fnbA (t = 4.833, P = 0.031) in the △SasX were significantly lower than those in the wild strain. The levels of icaA (t = 4.386, P = 0.034) and sarA (t = 5.531, P = 0.023) mRNA in the △SasX (pRB473-SasX) + RIP strain were significantly lower than those in the △SasX (pRB473-SasX). Under the microscope, it can be seen that the wild strains had large aggregates and clusters, and the △SasX were mostly scattered alone or aggregated in a small area. The aggregation ability of the △SasX was obviously weaker than that of the wild strain, the aggregation ability of the △SasX (pRB473-SasX) + RIP strain was weaker than that of the △SasX (pRB473-SasX).

SA SasX promotes the aggregation and enhances the ability of BF formation of SA by regulating the transcriptional expression of RNAⅢ, and thus participates in its pathogenic processes.