OmpK35/36 modulate the virulence of hypervirulent Klebsiella pneumoniae by regulating capsular polysaccharide export

This study aimed to investigate the impact of outer membrane proteins 35 (OmpK35) and 36 on the virulence of Klebsiella pneumoniae and the corresponding mechanisms. Serotype K1 strain NTUH-K2044 was utilized to create single-deletion mutants (Δ ompK35 and Δ ompK36 ) and a double-deletion mutant (Δ ompK35/36 ), alongside their complementation variants. Multiple analyses were performed, including capsule staining, transmission electron microscopy, serum resistance assays, phagocytosis tests, and infection models using mice and Galleria mellonella . The mutants retained hypercapsules similar to NTUH-K2044, but looser capsule structures revealed by transmission electron microscopy. All mutants exhibited decreased virulence, with median lethal doses under 5 × 10⁴ colony forming units in mice. Specifically, only Δ ompK35/36 showed reduced serum resistance compared to the wild strain, similar for the anti- phagocytosis capabilities. Additionally, Δ ompK35 induced liver inflammation, whereas Δ ompK36 and Δ ompK35/36 exerted milder effects. Despite these differences, all mutants displayed normal growth rates and upregulated expression of gnd and wcaJ , crucial for capsular polysaccharide (CPS) biosynthesis. The hypercapsule, composed mainly of CPS, plays a crucial role in the hypervirulence of K. pneumoniae . OmpK35/36 may modulate the virulence of K. pneumoniae by regulating CPS export.