Fig. 3.

K. pneumoniae O2 LPS is less immunogenic than K. pneumoniae O1 LPS. Purified O1 and O2 LPS were used for immunization of mice (a) or rabbits (b) with 0.5 mg LPS per animal. Immune sera were collected from O1 and O2 immunized animals and used to assess antibody titer against KP O1 or O2 LPS by ELISA. Sera from mock immunized animals with adjuvant only (black open circle) were used as a negative control. c Mice were immunized subcutaneously with either heat killed Kp1131115 (WT, an O1 strain) or the isogenic matched Kp1131115ΔwbbYZ (ΔwbbYZ, O2 serotype). Sera were collected 7 days after the final injection and used to assess antibody titer against K. pneumoniae O1 or O2 LPS by ELISA. d Sera collected in c were used to assess antibody binding to a protein target (OmpA) and to proteinase K bacterial lysate by ELISA. e Immune activation of cells in vitro was compared for purified O1 and O2 LPS. RAW264.7 cells stably transfected with a NF-κB driven luciferase promoter were stimulated with various doses of K. pneumoniae O1 or K. pneumoniae O2 LPS for 2 h. Percent activation was calculated as the amount of luminescence in stimulated cells vs. non-stimulated. Error bars indicate s.d. of each data point. f A schematic representation of KP O1 and O2 LPS. The gray region represents the membrane proximal LPS core region, the black region represents the d-galactan I (d-gal I) repeating O antigen sugars, the hatched gray filled region represents the O1 specific d-galactan II (d-gal II) repeating O antigen sugars encoded by the wbbYZ locus. Deletion of this locus converted the O1 strain (Kp1131115 WT) to an O2 expressing strain (Kp1131115ΔwbbYZ). g Silver stain of a SDS-PAGE gel showing the relative molecular weight of K. pneumoniae O1 LPS (lane 2) vs. K. pneumoniae O2 LPS (lane 3)