Pneumococcal 6-phosphogluconate-dehydrogenase, a putative adhesin, induces protective immune response in mice

Abstract

For most bacteria, adherence to human cells is achieved by bacterial lectins binding to mammalian surface glyconjugates. 6-Phosphogluconate dehydrogenase (6PGD) was identified by us as one of Streptococcus pneumoniae cell wall lectin proteins, which elicits an age-dependent immune response in humans. This study assesses the role of 6PGD in S. pneumoniae pathogenesis as an adhesin and its ability to elicit a protective immune response in mice. Recombinant 6PGD (r6PGD) was cloned from S. pneumoniae serotype 3 (strain WU2). r6PGD interference in adhesion of three genetically unrelated unencapsulated pneumococcal strains (3.8, 14.8 and R6) and two genetically unrelated encapsulated pneumococcal strains (WU2 and D39) to A549 type II lung carcinoma cell was tested. BALB/c mice were immunized with r6PGD and boosted after 3 weeks. Immunized mice were challenged intranasally with a lethal dose of S. pneumoniae. r6PGD inhibited 90% and 80% of pneumococcal adhesion to the A549 cells of three unencapsulated S. pneumoniae strains and two encapsulated S. pneumoniae strains, respectively, in a concentration-dependent manner (P < 0.05). Antibodies to r6PGD produced in mice significantly inhibited bacterial adhesion to A549 cell (P < 0.05). Immunization of mice with r6PGD protected 60% (P < 0.001) of mice for 5 days and 40% (P < 0.05) of the mice for 21 days following intranasal lethal challenge. We have identified 6PGD as a surface-located immunogenic lectin protein capable of acting as an adhesin. 6PGD importance to bacterial pathogenesis was demonstrated by the ability of r6PGD to elicit a protective immune response in mice.

Fig. 1

Inhibition of unencapsulated Streptococcus pneumoniae adhesion to A549 epithelial cells. S. pneumoniae strains were added to the cultured A459 type II lung carcinoma cells prior and following treatment with increasing concentration of purified recombinant 6-phosphogluconate dehydrogenase (r6PGD). r6PGD interfered with the adhesion of unencapsulated S. pneumoniae strains, 3·8 (a), 14·8 (b) and R6 (c) in a concentration-dependent manner (3·8; r = −0·754, P < 0·001, 14·8; r = −0·626, P < 0·05, R6; r = −0·782, P < 0·001). Insert in each panel: primary fluorescence activated cell sorter (FACS) histogram overlays showing staining of the corresponding bacteria with anti-6PGD serum (plain line) and control mouse serum (dotted line).

Fig. 2

Inhibition of encapsulated Streptococcus pneumoniae adhesion to A549 epithelial cells. S. pneumoniae encapsulated strains WU2 (a) and D39 (b) were added to the cultured epithelial cells prior and following treatment with purified recombinant 6-phosphogluconate dehydrogenase (r6PGD). r6PGD inhibited S. pneumoniae adhesion in concentration-dependent (WU2; r = −0·921, P < 0·001, D39; r = −0·771, P < 0·001).

Fig. 3

The specificity of the serum obtained from the immunized mice. The specificity of the antibodies produced in mice immunized with recombinant 6-phosphogluconate dehydrogenase (r6PGD) was tested by two-dimensional polyacrylamide gel electrophoresis (PAGE) Western blot of total cell wall proteins. (a) Two-dimensional PAGE, transferred onto nitrocellulose membrane and probed with serum obtained from mice before immunization with r6PGD protein. (b) Two-dimensional PAGE, transferred onto nitrocellulose membrane and probed with serum obtained from mice after immunization with r6PGD protein. (c) r6PGD and total cell wall proteins of Streptococcus pneumoniae strains R6, WU2, 6BR, 9VR, 14DW and 14R were separated on PAGE, transferred onto nitrocellulose membranes and probed with sera obtained from mice immunized with r6PGD protein.

Fig. 4

Inhibition of Streptococcus pneumoniae adhesion to A549 lung cells by anti-r6PGD antibodies. Antibodies to r6PGD were produced after immunization of mice with recombinant 6-phosphogluconate dehydrogenase (r6PGD) protein. S. pneumoniae (strain 3·8) bacteria were added to A549 cells in the presence of pre-immune mouse serum (control) or serum obtained from r6PGD immunized mice. The serum obtained from immunized mice inhibited S. pneumoniae adhesion in a concentration-dependent manner (r = −0·886, P < 0·05).

Fig. 5

Survival of 6-phosphogluconate dehydrogenase (6PGD) immunized mice following S. pneumoniae challenge. Six-week-old BALB/c female mice were immunized intraperitoneally with 25 µg of r6PGD (n = 29) on day 0 (primary immunization) and day 21 (booster). Control mice (n = 14) were sham-immunized with adjuvant. r6PGD protected 60% and 40% of the mice after 5 (P < 0·001) and 21 (P < 0.05) days, respectively, after intranasal lethal challenge with S. pneumoniae WU2.