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.