RES-Xre toxin-antitoxin locus knaAT maintains the stability of the virulence plasmid in Klebsiella pneumoniae .

Hypervirulent Klebsiella pneumoniae isolates have been increasingly reported worldwide , 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 K. pneumoniae . More than 3,000 TA loci in 2,000  K. pneumoniae 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 K. pneumoniae . Overexpression of the RES toxin KnaT, encoded by the virulence plasmid-carrying RES-Xre locus knaAT, halts the cell growth of K. pneumoniae and E. coli , whereas co-expression of the cognate Xre antitoxin KnaA neutralizes the toxicity of KnaT. knaA and knaT were co-transcribed, representing the characteristics of a type II TA module. The knaAT deletion mutation gradually lost its virulence plasmid in K. pneumoniae, whereas the stability of the plasmid in E. coli was enhanced by adding knaAT , which revealed that the knaAT operon maintained the genetic stability of the large virulence plasmid in K. pneumoniae . String tests and mouse lethality assays subsequently confirmed that a loss of the virulence plasmid resulted in reduced pathogenicity of K. pneumoniae . These findings provide important insights into the role of the RES-Xre TA pair in stabilizing virulence plasmids and disseminating virulence genes in K. pneumonia e.