Overcoming phagocytosis resistance of hypervirulent Klebsiella pneumoniae by directly targeting capsules

Abstract Capsular polysaccharides (CPS) are key virulence factors in Klebsiella pneumoniae and are closely associated with the K1 and K2 hypervirulent serotypes. Herein, we demonstrate that introducing nonspecific RNA (sgRNA) into K. pneumoniae 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 rmpADC operon, a key virulence determinant located on mobile chromosomal elements. Additionally, the mRNA expression of manC , which are chromosomal cps -related genes, was significantly repressed. In contrast, introduction of pET-sgRNA did not alter the mRNA expression of galF or wzi . The results revealed that capsule loss and reduced hypermucoviscosity in Kp-pET-sgRNA resulted from synergistic downregulation of both the rmpADC operon and manC . Loss of capsule synthesis and reduction of hypermucoviscosity in K. pneumoniae 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 K. pneumoniae . This study highlights a promising strategy for disarming hypervirulent K. pneumoniae by directly targeting its key virulence factors and provides novel insights into antibacterial therapeutic approaches against this clinically significant pathogen.