Fig. 4.

Cross-protection against IPD is mediated by the lipoproteins PrsA and MalX. (A and B) Validation of the results from the IP experiment and mass spectrometry analysis confirming the identity of the major immunoreactive proteins. Lysates from T4 WT; single mutants T4ΔpspA, T4ΔprsA, and T4ΔmalX; double mutants T4ΔpspAΔprsA and T4ΔpspAΔmalX; and triple mutant T4ΔpspAΔmalXΔprsA as indicated were subjected to Western blot analysis. PVDF membranes were incubated with immune sera from mice immunized with (A) T4 MP+Adjuvant or (B) T4ΔpspA MP+Adjuvant. Sera came from mice included in experiments presented in Fig. 3 C–E. A second incubation with sheep anti-mouse IgG–HRP conjugated facilitated chemiluminescent detection. (C–E) Percentage of mice immunized with either T4 MPs or T4ΔpspAΔmalXΔprsA MPs that survived an intranasal infection with (C) T4, (D) serotype 1 (BHN733), or (E) serotype 3 (BHN428) bacteria; 10 mice per group (the group immunized with T4ΔpspAΔmalXΔprsA MPs and infected with T4 included 9 mice). Five mice/pneumococcal strain were immunized with adjuvant as control. (F–H) CFUs in the lungs of mice infected with (F) T4, (G) serotype 1 (BHN733), or (H) serotype 3 (BHN428) bacteria at sacrifice. Dashed horizontal line in (F–H) represents the limit of detection for the lung CFU counts using this method. (I) Percentage of mice immunized with T4, T4ΔpspAΔmalX, T4ΔpspAΔprsA, T4ΔmalXΔprsA, orT4ΔpspAΔmalXΔprsA MPs that survived an intranasal infection with serotype 1 strain BHN733, showing both MalX and PrsA are required for high percentage of survival. (J) CFUs in the lungs of mice from the experiment in I; 10 mice per group (except for T4 MP + Adjuvant, which had 9 mice). Five mice/pneumococcal strain were immunized with adjuvant as control. Each dot represents one mouse. Error bars represent the standard error of the mean (SEM). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.