Streptococcus pneumoniae surface protein PcpA elicits protection against lung infection and fatal sepsis

Abstract

Previous studies have suggested that pneumococcal choline binding protein A (PcpA) is important for the full virulence of Streptococcus pneumoniae, and its amino acid sequence suggests that it may play a role in cellular adherence. PcpA is under the control of a manganese-dependent regulator and is only expressed at low manganese concentrations, similar to those found in the blood and lungs. PcpA expression is repressed under high manganese concentrations, similar to those found in secretions. In this study, we have demonstrated that PcpA elicits statistically significant protection in murine models of pneumonia and sepsis. In the model of pneumonia with each of four challenge strains, statistically fewer S. pneumoniae cells were recovered from the lungs of mice immunized with PcpA and alum versus mice immunized with alum only. The immunizations reduced the median CFU by 4- to 400-fold (average of 28-fold). In the model of sepsis using strain TIGR4, PcpA expression resulted in shorter times to become moribund and subcutaneous immunization with PcpA increased survival times of mice infected with wild-type PcpA-expressing pneumococci.

FIG. 1.

Western blot analysis of the presence of PcpA under low-manganese conditions. After bacteria were cultured in a low-manganese medium until mid-log phase, total cellular protein samples were prepared. Samples were separated by SDS-PAGE, transferred to nitrocellulose, and probed with an rPcpA polyclonal antiserum. Lane 1, TIGR4 pcpA (pcpA mutant); lane 2, TIGR4 psaR (pcpA constitutive mutant); lane 3, D1091B; lane 4, EF5668; lane 5, BG10752; lane 6, V175; lane 7, L82013; lane 8, BG12730; lane 9, TJ0893. When preimmune rabbit serum was used in place of immune rabbit sera, the PcpA bands were not detected (data not shown). When the same strains were cultured in Todd-Hewitt broth (high manganese) none of them produced detectable PcpA (data not shown).

FIG. 2.

Surface exposure of PcpA on S. pneumoniae strain TIGR4. TIGR4 was cultured in (A) high- or (B) low-manganese medium until mid-log phase. The bacteria were incubated with anti-PcpA rabbit serum, followed by incubation with FITC-conjugated anti-rabbit Ig antibodies. The cells were next fixed in 4% formaldehyde containing the membrane dye TMA-DPH. Blue fluorescence indicates staining with TMA-DPH, and green fluorescence indicates staining with anti-PcpA antibodies. (C) Bacterial culture in high-manganese medium, incubated with FITC-conjugated antibodies to rabbit Ig, without any rabbit anti-PcpA.

FIG. 3.

Protection conferred by rPcpA immunization in a murine model of pneumonia. CBA/N mice were subcutaneously immunized with rPcpA adsorbed to aluminum hydroxide or with aluminum hydroxide alone. The mice were challenged intranasally under light anesthesia with 5 × 10⁶ CFU of EF3030. After the mice were sacrificed 7 days postinfection, bacterial counts were determined from lung homogenates (A) and nasal washes (B). The horizontal line denotes median log₁₀ CFU. **, P = 0.0019, Mann-Whitney test.

FIG. 4.

Protection conferred against other S. pneumoniae capsular serotypes by rPcpA immunization in a murine model of pneumonia. Mice were challenged with the following strains: (A) TJ0893, serotype 14 (**, P = 0.0209); (B) L82016X, serotype 6B (**, P = 0.0193); and (C) EF9303, serotype 23F (**, P = 0.0388) (Mann-Whitney test). The horizontal line denotes median log₁₀ CFU.

FIG. 5.

Effect of pcpA inactivation on intranasal colonization of S. pneumoniae. The mice were challenged intranasally with 10⁶ CFU of EF3030 or EF3030 pcpA. After the mice were sacrificed 7 days postinfection, bacterial counts were determined from nasal washes. The horizontal line denotes median log₁₀ CFU/nose.

FIG. 6.

Protection conferred by rPcpA immunization in a murine model of sepsis. CBA/N mice were subcutaneously immunized with rPcpA adsorbed to aluminum hydroxide or aluminum hydroxide alone. After the mice were challenged intravenously with 300 CFU of TIGR4, their survival times were monitored for 21 days. The horizontal line denotes median survival time. **, P = 0.0067 (Mann-Whitney test).

FIG. 7.

Virulence of TIGR4 and TIGR4 pcpA in a murine model of sepsis. After the mice were challenged intravenously with 300 CFU of TIGR4 or TIGR4 pcpA, their survival times were monitored for 21 days. The horizontal line denotes median survival time. **, P = 0.0299 (Mann-Whitney test).