Sialic acid involved in hypermucoviscosity phenotype of Klebsiella pneumoniae and associated with resistance to neutrophil phagocytosis

Klebsiella pneumoniae (KP) with the hypermucoviscosity (HV) phenotype has abundant capsular polysaccharides (CPS) and usually causes an invasive syndrome. Sialic acid (Sia), a component of CPS in KP strains with the HV phenotype, may be anti-phagocytic. Sia-binding immunoglobulin-like lectin-9 (Siglec-9) act as an MHC class-I receptor on neutrophils that recognizes Sia and sends a signal to dampen inflammatory response. Three clinical KP strains with KP-M1 (HV-positive; capsular serotype K₁), KP-14 (HV-negative; capsular serotype non-K₁/K₂), and DT-X (HV-negative; capsular serotype K₁) were studied. We assessed total Sia in CPS extracts using enzymatic methods and phagocytosis by neutrophils of neuraminidase-treated bacteria using flow cytometry. Neutrophil killing was evaluated in the presence and absence of antibodies against Siglec-9. The concentration of Sia was significantly higher in the CPS extract of KP-M1 (56.75 ± 6.75 μmole/10⁹ cfu) than in the CPS extract of KP-14 (0.02 ± 0.01 μmole/10⁹ cfu) and DT-X (a negligible value). The KP-M1 (compared with the KP-14 and DT-X) was more resistant to neutrophil phagocytosis. Both the HV phenotype and resistance to phagocytosis of KP-M1 were significantly decreased after Sia removal with neuraminidase treatment. Fluorescence microscopy with an antibody against human Siglec-9 showed attachment of KP-M1 (but were absent of KP-14 and DT-X) to the surface of neutrophils and colocalization with human Siglec-9. Engagement of Siglec-9 via Sia enhanced neutrophils killing of KP-M1 by ex vivo human neutrophils bactericidal activity assay. The result showed that Sia might be a constituent of KP-M1 CPS responsible for HV, thereby contributing to anti-phagocytic activity of this pathogen.