Role of pneumococcal surface protein C in nasopharyngeal carriage and pneumonia and its ability to elicit protection against carriage of Streptococcus pneumoniae

Abstract

Previous studies suggested that PspC is important in adherence and colonization within the nasopharynx. In this study, we conducted mutational studies to further identify the role PspC plays in the pathogenesis of pneumococci. pspC and/or pspA was insertionally inactivated in a serotype 2 Streptococcus pneumoniae strain and in a serotype 19 S. pneumoniae strain. In the mouse colonization model, pneumococcal strains with mutations in pspC were significantly attenuated in their abilities to colonize. In a mouse pneumonia model, strains with mutations in pspC were unable to infect or multiply within the lung. Using reverse transcriptase PCR we were able to demonstrate that pspC is actively transcribed in vivo, when the bacteria are growing in the nasal cavity and in the lungs. In the bacteremia model, a strain mutated for pspC alone behaved like the wild type, but the absence of both pspC and pspA caused accelerated clearance of the bacteria. Intranasal immunization with PspC with cholera toxin subunit B as an adjuvant protected against intranasal challenge. Evidence was also obtained that revertants that spontaneously acquired PspC expression could multiply and colonize the nasal tissue. This latter finding strongly indicates that pneumococci are actively metabolizing and growing while in the nasopharynx.

FIG. 1.

Southern blots of chromosomal DNA from D39 transformants. Chromosomal DNA was run in duplicate and developed with a pspC-specific probe (A) or with a full-length pspA probe (B). The DNA fragment containing a mutated gene is shifted in strains where the plasmid has been inserted. An asterisk indicates the fragment containing the pspC gene.

FIG. 2.

Effect of mutation of pspA and pspC on intranasal carriage of D39. CBA/N mice were infected with 1 × 10⁷ CFU of D39 or its isogenic mutants. Mice were sacrificed 7 days postinfection, and their nasal cavities were washed with 50 μl of Ringer's solution. The nasal washes were plated to enumerate the bacteria. P values versus D39 were calculated by using the Wilcoxon two-sample rank test. S.E., standard error.

FIG. 3.

Effect of pspC mutations on lung infection in CBA/N mice. Groups of six anesthetized CBA/N mice were challenged intranasally with 5 × 10⁶ CFU of wild-type EF3030 and two isogenic PspC-negative strains. Mice were sacrificed 6 days after challenge, and nasal wash, lung tissue, and blood samples were plated on blood agar plates to determine the number of bacteria.

FIG. 4.

Effect of mutations in pspC and pspA in a mouse bacteremia model. Groups of five mice (BALB/cByJ) were challenged intravenously with 1 × 10⁶ CFU of wild-type pneumococci (D39), pspA-negative D39 (JY53), pspC-negative D39 (TRE108), and pspA- and pspC-negative D39 (TRE121). The presence of bacteria in the blood was determined at 1 min, 1 h, and 4 h (a). Subsequently, the number of hours to death was determined in each case (b). P values versus the wild type were calculated by using the Wilcoxon two-sample rank test. Mice that did not die after 21 days (504 h) were assigned a time to death of 504 h for statistical purposes. In each graph, values that are significantly different from D39 are indicated by asterisks.

FIG. 5.

Detection of transcripts by RT-PCR. RNA was extracted from strain D39 in the nasal washes (a) or from strain EF3030 in the nasal washes and lung homogenates (b) of CBA/N mice. RNA was treated with DNase I. Transcripts were detected by RT-PCR with gene-specific primers. Transcripts detected were for pspC, endA, and pspA (a) and pspC (b). The plus and minus signs in panel b indicate reactions carried out in the presence (test) or absence (control) of RT, respectively. Similar controls were also used for the experiments whose results are shown in panel a.

FIG. 6.

Intranasal immunization with PspC. CBA/N mice were immunized with 2 μg of purified PspC and 4 μg of CTB or CTB alone once a week for 3 weeks. Three weeks after the last immunization, the mice were challenged intranasally with PLN-A (pneumolysin mutant of D39). Mice were sacrificed after 7 days, the nasal cavities were washed with Ringer's solution, and the nasal wash samples were plated to determine the CFU counts. P values were calculated by using the one-tailed Wilcoxon test. One mouse in the CTB group died and was assigned a nasal wash log CFU value of 6 for statistical purposes.