Small regulatory RNA RSaX28 promotes virulence by reinforcing the stability of RNAIII in community-associated ST398 clonotype <i>Staphylococcus aureus</i>

<i>Staphylococcus aureus</i> (<i>S. aureus</i>) is a notorious pathogen that cause metastatic or complicated infections. Hypervirulent ST398 clonotype strains, remarkably increased in recent years, dominated Community-associated <i>S. aureus</i> (CA-SA) infections in the past decade in China. Small RNAs like RNAIII have been demonstrated to play important roles in regulating the virulence of <i>S. aureus</i>, however, the regulatory roles played by many of these sRNAs in the ST398 clonotype strains are still unclear. Through transcriptome screening and combined with knockout phenotype analysis, we have identified a highly transcribed sRNA, RSaX28, in the ST398 clonotype strains. Sequence analysis revealed that RSaX28 is highly conserved in the most epidemic clonotypes of <i>S. aureus</i>, but its high transcription level is particularly prominent in the ST398 clonotype strains. Characterization of RSaX28 through RACE and Northern blot revealed its length to be 533nt. RSaX28 is capable of promoting the hemolytic ability, reducing biofilm formation capacity, and enhancing virulence of <i>S. aureus</i> in the <i>in vivo</i> murine infection model. Through IntaRNA prediction and EMSA validation, we found that RSaX28 can specifically interact with RNAIII, promoting its stability and positively regulating the translation of downstream alpha-toxin while inhibiting the translation of Sbi, thereby regulating the virulence and biofilm formation capacity of the ST398 clonotype strains. RSaX28 is an important virulence regulatory factor in the ST398 clonotype <i>S. aureus</i> and represents a potential important target for future treatment and immune intervention against <i>S. aureus</i> infections.金黄色葡萄球菌（Staphylococcus aureus，简称S. aureus）是一种可引发转移性或复杂性感染的高致病性病原菌。近年来数量显著攀升的高毒力ST398克隆型菌株，在过去十年间主导了中国境内的社区获得性金黄色葡萄球菌（Community-associated S. aureus, CA-SA）感染。诸如RNAIII的小RNA（small RNAs, sRNAs）已被证实可在调控金黄色葡萄球菌的毒力过程中发挥重要作用，但目前仍不清楚多数此类小RNA在ST398克隆型菌株中所扮演的调控角色。本研究通过转录组筛选结合敲除表型分析，在ST398克隆型菌株中鉴定出一种高转录水平的小RNA RSaX28。序列分析结果显示，RSaX28在金黄色葡萄球菌的多数流行克隆型中均呈现高度保守性，但其高转录水平在ST398克隆型菌株中尤为显著。通过快速扩增cDNA末端（rapid amplification of cDNA ends, RACE）与Northern印迹杂交（Northern blot）对RSaX28进行表征，结果显示其长度为533 nt（核苷酸, nt）。在体内（in vivo）小鼠感染模型中，RSaX28可增强金黄色葡萄球菌的溶血能力，降低其生物膜形成能力，并提升其毒力。通过IntaRNA预测与电泳迁移率变动分析（electrophoretic mobility shift assay, EMSA）验证，本研究发现RSaX28可与RNAIII发生特异性结合，促进RNAIII的稳定性，正向调控下游α毒素的翻译并抑制Sbi的翻译，进而调控ST398克隆型菌株的毒力与生物膜形成能力。RSaX28是ST398克隆型金黄色葡萄球菌中重要的毒力调控因子，同时也是未来针对金黄色葡萄球菌感染开展治疗与免疫干预的潜在重要靶点。