RES-Xre toxin-antitoxin locus <i>knaAT</i> maintains the stability of the virulence plasmid in <i>Klebsiella pneumoniae</i>

Hypervirulent <i>Klebsiella pneumoniae</i> isolates have been increasingly reported worldwide<i>,</i> especially hypervirulent drug-resistant variants owing to the acquisition of a mobilizable virulence plasmid by a carbapenem-resistant strain. This pLVPK-like mobilizable plasmid encodes various virulence factors; however, information about its genetic stability is lacking. This study aimed to investigate the type II toxin-antitoxin (TA) modules that facilitate the virulence plasmid to remain stable in <i>K. pneumoniae</i>. More than 3,000 TA loci in 2,000 <i>K. pneumoniae</i> plasmids were examined for their relationship with plasmid cargo genes. TA loci from the RES-Xre family were highly correlated with virulence plasmids of hypervirulent <i>K. pneumoniae</i>. Overexpression of the RES toxin KnaT, encoded by the virulence plasmid-carrying RES-Xre locus <i>knaAT,</i> halts the cell growth of <i>K. pneumoniae</i> and <i>E. coli</i>, whereas co-expression of the cognate Xre antitoxin KnaA neutralizes the toxicity of KnaT. <i>knaA</i> and <i>knaT</i> were co-transcribed, representing the characteristics of a type II TA module. The <i>knaAT</i> deletion mutation gradually lost its virulence plasmid in <i>K. pneumoniae,</i> whereas the stability of the plasmid in <i>E. coli</i> was enhanced by adding <i>knaAT</i>, which revealed that the <i>knaAT</i> operon maintained the genetic stability of the large virulence plasmid in <i>K. pneumoniae</i>. String tests and mouse lethality assays subsequently confirmed that a loss of the virulence plasmid resulted in reduced pathogenicity of <i>K. pneumoniae</i>. These findings provide important insights into the role of the RES-Xre TA pair in stabilizing virulence plasmids and disseminating virulence genes in <i>K. pneumonia</i>e.高毒力肺炎克雷伯菌（Hypervirulent Klebsiella pneumoniae）分离株在全球范围内的报道日益增多，尤其是碳青霉烯耐药菌株通过获得可移动毒力质粒而演化出的高毒力耐药变体。这类类似pLVPK的可移动质粒可编码多种毒力因子，但目前关于其遗传稳定性的研究信息仍较为匮乏。本研究旨在探究可帮助肺炎克雷伯菌维持毒力质粒稳定性的II型毒素-抗毒素（toxin-antitoxin, TA）模块。本研究对2000株肺炎克雷伯菌质粒中的3000余个TA基因座进行了分析，以探究其与质粒载荷基因的关联。结果显示，属于RES-Xre家族的TA基因座与高毒力肺炎克雷伯菌的毒力质粒呈现高度相关性。由携带毒力质粒的RES-Xre基因座knaAT编码的RES家族毒素KnaT过表达时，可抑制肺炎克雷伯菌与大肠埃希菌（E. coli）的细胞增殖；而其同源Xre家族抗毒素KnaA的共表达则可中和KnaT的毒性。knaA与knaT共转录，符合II型TA模块的典型特征。在肺炎克雷伯菌中，敲除knaAT的突变株会逐渐丢失其携带的毒力质粒；而在大肠埃希菌中引入knaAT则可增强其质粒的稳定性，这表明knaAT操纵子可维持肺炎克雷伯菌中大毒力质粒的遗传稳定性。随后通过拉丝试验与小鼠致死性实验证实，毒力质粒的丢失会降低肺炎克雷伯菌的致病力。本研究结果为解析RES-Xre TA系统对肺炎克雷伯菌毒力质粒的稳定作用以及毒力基因的传播机制提供了重要参考。