The Role Of Pneumolysin Sequence Variants And Strain Background In The Virulence Of Streptococcus Pneumoniae

Streptococcus pneumoniae is a major cause of pneumonia, bacteraemia, meningitis and otitis media. Pneumolysin (Ply) is the key toxin of the bacterium, and it has many adverse effects on immune cell functions. Ply variants have been described among clinical pneumococcal. These variants were reported to differences in cytotoxic activity but the impact of these differences on virulence is hard to ascertain because they are being made in different strain background. To study the significant of polymorphisms of Ply function in a single genetic background, unmarked mutations were introduced into the ply in the S. pneumoniae type 2 D39 strain background using pORI280 to replace the natural copy of ply gene. The selected Ply variants were expressed and purified using affinity chromatography and gel filtration to determine haemolytic and complement activation activities. The virulence of the D39 recombinant carrying variants of ply were tested in a mouse model of pneumococcal pneumonia. The results showed that of eight naturally occurring single nucleotide polymorphisms (SNP), all Ply alleles reduced the haemolytic activity of Ply in different degrees compared to the D39 Ply excepting the Ply allele 9 which increased haemolytic activity. In vivo, D39 expressing Ply alleles 2, 8, 11, 12 or 18 are significantly attenuated whereas Ply alleles 9 and 15 were not attenuated in virulence in a pneumonia model of infection. The numbers of CFU for streptococcus carrying ply alleles 2, 8, 11, 12 and 18 were significantly lower in the lung and spleen tissues, compared with wild type D39. Furthermore, pneumococcal bacteraemia in mice infected with D39 expressing Ply alleles 2, 8, 11, 12 or18 were significantly attenuated compared with wild type D39. In conclusion the Ply variations and the genetic background of the S. pneumoniae strain carrying them have an important impacts on pneumococcal virulence.