Polysaccharide capsule-mediated resistance to opsonophagocytosis in Klebsiella pneumoniae

Abstract

The polysaccharide capsule of Klebsiella pneumoniae is an important virulence factor that confers resistance to phagocytosis. The treatment of encapsulated bacteria with salicylate to inhibit capsule expression was found to enhance the phagocytosis of encapsulated bacteria by human neutrophils only in the presence of cell surface-specific antibodies. Both type-specific rabbit antisera and anticapsular human hyperimmune globulin were employed as opsonins. Salicylate significantly enhanced phagocytosis with homologous, but not heterologous, whole-cell antisera. Antisera, diluted 1:40, no longer opsonized fully encapsulated bacteria but promoted the uptake of multiple salicylate-treated bacteria in > 90% of neutrophils. Salicylate (0.25 to 1.0 mM) also enhanced opsonization with globulin against homologous bacteria. Higher salicylate levels (1 to 2.5 mM) enhanced the opsonization of heterologous serotypes with human globulin. The nature of antibody attachment to encapsulated bacteria was determined by immunofluorescence. Even after the addition of purified capsular polysaccharide to prevent phagocytosis, K-specific antibodies attached in large amounts to bacteria. K-specific antibodies reacted with antigens throughout the capsule and showed a predilection for a denser inner layer of the capsule, indicating that many of the K-specific antibodies may be masked underneath the capsule surface. K-specific antibodies can also be rendered nonfunctional by soluble, cell-free capsular antigen. In culture, large quantities of soluble capsular polysaccharide extrude from bacteria after overnight growth. The reduction in capsule expression caused by salicylate largely affected the soluble, cell-free fraction. Purified capsular polysaccharide was shown to retard the opsonophagocytosis of salicylate-treated bacteria in a concentration-dependent manner. However, extensive washing of encapsulated bacteria to remove loosely attached capsular material did not significantly enhance opsonophagocytosis. In conclusion, cell-free capsule and cell-associated capsule are antiphagocytic; both act to neutralize K-specific antibodies by binding or concealment. Salicylate-mediated inhibition of capsule expression, particularly of the cell-free fraction, improved K-specific opsonization dramatically.