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Previous work has demonstrated the capsular serotypic specificity of the protection conferred on mice by Klebsiella pneumoniae ribosomal preparations. The data in these studies support the hypothesis that capsular polysaccharide plays at least some role in the specificity of the protection conferred by ribosomal preparations. In this investigation, the presence of capsular polysaccharide and lipopolysaccharide in K. pneumoniae ribosomal preparations was demonstrated by using immunodiffusion tests. Lipopolysaccharide content was determined for mice treated with actinomycin D. The serotype of O antigen did not play a role in the orientation of the specificity of the protection. The possibility that lipopolysaccharide might act as an adjuvant was not unlikely since the ribosomal preparations which contained the greatest amounts of lipopolysaccharide appeared to be the most immunoprotective preparations. Ribosomal preparations extracted from a noncapsulated mutant of K. pneumoniae did not protect mice. This finding suggested that capsular polysaccharide might play a role in the immunoprotective activity of ribosomes. This hypothesis was tested by using K. pneumoniae K2 bacteriophage-associated-glycanase, which specifically hydrolyzed K. pneumoniae K2 capsular polysaccharide and thereby suppressed the immunoprotective activity of K. pneumoniae K2 ribosomal preparations. In contrast, the K2 bacteriophage-associated glycanase did not interfere with the immunoprotective activity of K. pneumoniae K1 ribosomal preparations. These results clearly demonstrate that capsular polysaccharide, which is an extraribosomal antigen, is involved in the immunoprotective activity of K. pneumoniae ribosomal preparations.

Two peaks were obtained by cesium chloride density gradient ultracentrifugation of Klebsiella pneumoniae ribosomal preparations. Peak I contained capsular polysaccharide, lipopolysaccharide, protein, and less than 0.5% ribonucleic acid. Peak II consisted mainly of ribonucleic acid, with low amounts of protein and capsular polysaccharide. Expressed as capsular polysaccharide content, the 50% protective dose of peak I and of nonfractionated ribosomal preparations was nearly constant (2.6 and 1.2 ng, respectively). Since peak I contained less than 0.5% ribonucleic acid, these results provide evidence that ribosomal ribonucleic acid is not required for protection of mice by K. pneumoniae capsular polysaccharide which contaminates ribosomal preparations.

Cell surface preparations and ribosomal preparations were extracted from Klebsiella pneumoniae. Agar gel diffusion with antisera to cell surface preparations or ribosomal preparations indicated common antigenic components among the preparations. Lipopolysaccharide and capsular polysaccharide were identified in the cell surface preparations. These results and the previous identification of lipopolysaccharide and capsular polysaccharide in ribosomal preparations suggest that these antigens are responsible for the immunochemical cross-reactivity observed among these two bacterial extracts. Active protection could be induced in mice by these two preparations. On a dry-weight basis, cell surface preparations provided better immunoprotective activity than did ribosomal preparations. However, the 50% protective dose of both preparations is practically the same on the basis of their capsular polysaccharide content. These results are consistent with the hypothesis that the immunoprotective moiety of ribosomal preparations is the contaminating cell surface antigens. Furthermore, the low level of nucleotidic components detected in purified cell surface preparations led us to infer that the immunoprotective activity of capsular polysaccharide may not be dependent on the adjuvant activity of ribonucleic acid. The involvement of capsular polysaccharide in the immunoprotective capacity of cell surface preparations is demonstrated either by using a degradation of this antigen by K. pneumoniae bacteriophage K2-associated glycanase or by using a preparation extracted from a noncapsulated mutant of K. pneumoniae. Nevertheless, the low protective ability of purified capsular polysaccharides is in contrast to its greater activity when induced in bacterial cell surface preparations. The protective activity of K. pneumoniae capsular polysaccharide may be dependent on its association with other surface antigenic components present in cell surface preparations or may be dependent on its native form in the bacterial cell surface.