Overcoming phagocytosis resistance of hypervirulent Klebsiella pneumoniae by directly targeting capsules

Capsular polysaccharides (CPS) are key virulence factors in <i>Klebsiella pneumoniae</i> and are closely associated with the K1 and K2 hypervirulent serotypes. Herein, we demonstrate that introducing nonspecific RNA (sgRNA) into <i>K. pneumoniae</i> ATCC43816 (Kp-pET-sgRNA), a K2 serotype strain classified as hypervirulent (hvKp), results in marked capsule loss and reduced hypermucoviscosity. Capsule loss and reduced hypermucoviscosity in Kp-pET-sgRNA were confirmed by comparison with the wild-type strain (Kp-WT) using transmission electron microscopy, hypermucoviscosity assays, and string tests. Mechanistically, we found that overexpression of sgRNA by introducing the pET-sgRNA plasmid led to gene deletion in the <i>rmpADC</i> operon, a key virulence determinant located on mobile chromosomal elements. Additionally, the mRNA expression of <i>manC</i>, which are chromosomal <i>cps</i>-related genes, was significantly repressed. In contrast, introduction of pET-sgRNA did not alter the mRNA expression of <i>galF</i> or <i>wzi</i>. The results revealed that capsule loss and reduced hypermucoviscosity in Kp-pET-sgRNA resulted from synergistic downregulation of both the <i>rmpADC</i> operon and <i>manC</i>. Loss of capsule synthesis and reduction of hypermucoviscosity in <i>K. pneumoniae</i> caused by sgRNA overexpression significantly decreased resistance to phagocytosis by macrophages but did not influence susceptibility to meropenem or colistin. The findings reveal an unexpected consequence of plasmid-mediated sgRNA introduction, where overexpression of sgRNA abolishes phagocytic resistance by disrupting capsule biosynthesis and reducing hypermucoviscosity in <i>K. pneumoniae</i>. This study highlights a promising strategy for disarming hypervirulent <i>K. pneumoniae</i> by directly targeting its key virulence factors and provides novel insights into antibacterial therapeutic approaches against this clinically significant pathogen.