The three extra-cellular zinc metalloproteinases of Streptococcus pneumoniae have a different impact on virulence in mice

Abstract

Background: Streptococcus pneumoniae possesses large zinc metalloproteinases on its surface. To analyse the importance in virulence of three of these metalloproteinases, intranasal challenge of MF1 outbred mice was carried out using a range of infecting doses of wild type and knock-out pneumococcal mutant strains, in order to compare mice survival.

Results: Observation of survival percentages over time and detection of LD50s of knock out mutants in the proteinase genes in comparison to the type 4 TIGR4 wild type strain revealed two major aspects: i) Iga and ZmpB, present in all strains of S. pneumoniae, strongly contribute to virulence in mice; (ii) ZmpC, only present in about 25% of pneumococcal strains, has a lower influence on virulence in mice.

Conclusions: These data suggest Iga, ZmpB and ZmpC as candidate surface proteins responsible for pneumococcal infection and potentially involved in distinct stages of pneumococcal disease.

Figure 1

Unrooted radial tree of streptococcal zinc metalloproteinases: The tree diagram of streptococcal zinc metalloproteinases was drawn to visualise to which group the diverse proteinases of strain TIGR4 belong to. The major clusters formed by the zinc metalloproteinases of the oral streptococci are circled. The three enzymes of strain TIGR4, whose deletion mutants are assayed in this work, are boxed. Even if streptococcal zinc metalloproteinases evolve by horizontal gene transfer and show evident mosaic like structure, a fact questioning phylogenic analysis, a tree was drawn to show the major grouping of these enzymes. A stretch of 180 amino acids surrounding the active site of the streptococcal zinc metalloproteinases available from GenBank [5-8], TIGR and Sanger Centre were aligned with ClustalX using standard parameters and the tree was visualised using TreeView. S. pneumoniae proteins are preceded by Sp and the serotype. Proteins of the unfinished genomes of serotype 6 (TIGR) and 23F (Sanger Centre) are named as homologs referring to the published pneumococcal enzymes. The proteins derived from the unfinished genomes of S. gordonii and S. mitis (both TIGR) are denominated according to the respective contigs (last accessed January 27^th 2003).

Figure 2

Virulence of pneumococcal zinc metalloproteinase mutants at different doses of intranasal challenge: Mice were infected intranasally (i.n.) with different doses: panel (A) 10⁷ CFU/mouse, panel (B) 10⁶ CFU/mouse, and panel (C) 10⁵ CFU/mouse. Bacterial strains were TIGR4 (open circles, n = 12 for 10⁶ CFU and n = 6 for 10⁷ and 10⁵ CFU), ΔzmpC (solid triangles, n = 12 for 10⁶ CFU and n = 6 for 10⁷ and 10⁵ CFU), ΔzmpB (open diamonds, n = 6) and Δiga (crosses, n = 6) mutants. Animals were observed for 8 days. Results are expressed as percentage of mice surviving intranasal challenge and subsequent sepsis over time. Statistical analysis (Mann-Whitney-Wilcoxon U Test) was performed considering the time point when mice died (P < 0.01). For statistical purposes, animals still alive after 8 days were assigned a time to death of 192 h. The data referring to the 10⁶ CFU inoculum of the ΔzmpC mutant were already reported in reference [5] combined to bacteremia levels.

Figure 3

LD50s of mice challenged intranasally with pneumococcal zinc metalloproteinase mutants: (A) Six groups of MF1 outbred mice (n = 12) were infected i.n. with different inocula (10⁵, 10⁶, and 10⁷ CFU) of either TIGR4 wild type strain (open circles) or zmpC mutant (solid circles). (B) Six groups of MF1 outbred mice (n = 6) were inoculated i.n. with 10⁵,10⁶ and 10⁷ CFU of TIGR4 (open circles) or zmpB mutant (solid triangles). (C) Six groups of MF1 outbred mice (n = 6) were infected with different bacterial doses (10⁵, 10⁶, and 10⁷ CFU) of TIGR4 (open circles) or iga mutant (solid diamonds). LD₅₀s are indicated by the dotted line. Differences in survival percentages between the parent strain and IgA protease mutants were statistically analyzed by Fisher's exact test (P < 0.05). LD₅₀ calculation was performed 8 days after observation of survival. Data reported in this figure are taken from the same experiment as those in figure 2.