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.