Additive attenuation of virulence of Streptococcus pneumoniae by mutation of the genes encoding pneumolysin and other putative pneumococcal virulence proteins

Abstract

Although the polysaccharide capsule of Streptococcus pneumoniae has been recognized as a sine qua non of virulence, much recent attention has focused on the role of pneumococcal proteins in pathogenesis, particularly in view of their potential as vaccine antigens. The individual contributions of pneumolysin (Ply), the major neuraminidase (NanA), autolysin (LytA), hyaluronidase (Hyl), pneumococcal surface protein A (PspA), and choline-binding protein A (CbpA) have been examined by specifically mutagenizing the respective genes in the pneumococcal chromosome and comparing the impact on virulence in a mouse intraperitoneal challenge model. Mutagenesis of either the ply, lytA, or pspA gene in S. pneumoniae D39 significantly reduced virulence, relative to that of the wild-type strain, indicating that the respective gene products contribute to pathogenesis. On the other hand, mutations in nanA, hyl, or cbpA had no significant impact. The virulence of D39 derivatives carrying a ply deletion mutation as well as an insertion-duplication mutation in one of the other genes was also examined. Mutagenesis of either nanA or lytA did not result in an additional attenuation of virulence in the ply deletion background. However, significant additive attenuation in virulence was observed for the strains with ply-hyl, ply-pspA, and ply-cbpA double mutations.

FIG. 1

Southern hybridization analysis of insertion-duplication mutants. Chromosomal DNA from the indicated S. pneumoniae derivatives was digested with HindIII (for lytA and nanA mutants), EcoRI (for cbpA and hyl mutants), or ClaI (for pspA mutants). Replicate digests were subjected to Southern hybridization analysis using probes specific for the respective virulence factor gene (lytA, cbpA, hyl, pspA, or nanA) and pVA891, as described in Materials and Methods. Lanes: M, prelabelled DNA size marker (bacteriophage SPP1 DNA restricted with EcoRI; sizes from top to bottom are 8.56, 7.43, 6.11, 4.90, 3.64, 2.80, 1.95, 1.88, 1.52, 1.41, and 1.16 kb); P, ΔPly; L1, LytA⁻; L2, ΔPly-LytA⁻; C1, CbpA⁻; C2, ΔPly-CbpA⁻; H1, Hyl⁻; H2, ΔPly-Hyl⁻; P1, PspA⁻; P2, ΔPly-PspA⁻; N1, NanA⁻; N2, ΔPly-NanA⁻.

FIG. 2

Western immunoblot analysis. Lysates of the indicated S. pneumoniae D39 derivatives were separated by SDS-PAGE, electroblotted, and probed with mouse anti-PspA or mouse anti-LytA, as described in the Materials and Methods. Lanes: D, D39; P1, PspA⁻; P2, ΔPly-PspA⁻; Ply, ΔPly; L1, LytA⁻; L2, ΔPly-LytA⁻. The mobilities of protein size markers are also indicated.

FIG. 3

Survival times of mice after i.p. challenge. Groups of 12 or 13 BALB/c mice were injected i.p. with approximately 10³ CFU of the indicated strains. Each datum point represents one mouse. The horizontal lines denote the median survival time for each group.

FIG. 4

Survival times of mice after i.p. challenge. Groups of 12 BALB/c mice were injected i.p. with approximately 10⁵ CFU of the indicated strains. Each datum point represents one mouse. The horizontal lines denote the median survival time for each group.

FIG. 5

Survival times of mice after i.p. challenge. Groups of 12 BALB/c mice were injected i.p. with approximately 5 × 10³ CFU of the indicated strains. Each datum point represents one mouse. The horizontal lines denote the median survival time for each group.

FIG. 6

Survival times of mice after i.p. challenge. Groups of 12 BALB/c mice were injected i.p. with approximately 8 × 10⁶ CFU of the indicated strains. Each datum point represents one mouse. The horizontal lines denote the median survival time for each group.

FIG. 7

Survival times of mice after intranasal challenge. Groups of 12 QS mice were anesthetized and challenged intranasally with approximately 5 × 10⁶ CFU of the indicated strains. Each datum point represents one mouse. The horizontal lines denote the median survival time for each group.