Characterization of virulence in clinical isolates of Streptococcus pneumoniae

The immune system together with competing pneumococcal strains and other bacterial species, impose a selective pressure on the pneumococcus that leads to the expansion of certain lineages and the remission of others. Our results show that the invasive disease potential varies between serotypes, and also between sequence types (STs) determined by Multi-Locus Sequence Typing (MLST). Furthermore, using Pulsed-Field Gel Electrophoresis (PFGE) we found a divergence of lineages within MLST clones, which was associated with differences in their potential to cause invasive disease. These PFGE clones show intraclonal variation in virulence factors, and in the ability to recruit factor H, a host complement factor utilized by the bacterium to evade the immune system.

Selection of pneumococcal variants occurs both during colonization and disease. We show that during invasive disease there was a selection for serotype 1 variants with impaired hydrogen peroxide production due to mutations in the pyruvate oxidase gene spxB. These variants showed greater virulence in vivo and were more resistant to clearance by macrophages. An examination of patient samples revealed that spxB mutants were present during invasive disease in humans as well.

Variations between pneumococcal strains also affect tissue tropisms during infection, as some serotypes are associated with pneumonia and others with invasive disease. Using two virulent strains of serotypes 2 and 3 we show that the manifestations of disease in mice differed. Infections caused by serotype 3 were primarily confined to the lungs, and the bacteria were resilient to opsonization and clearance by macrophages. In contrast, infection with serotype 2 caused an invasive infection, which spread rapidly from the lungs to the blood.

The genome of the pneumococcus, including several of the bacterial strains in this thesis, has been shaped by the incorporation of lysogenic bacteriophages. We show that a bacteriophage contributed to the virulence during invasive disease of a serotype 1 strain, a serotype with high invasiveness. In the absence of the phage encoded pblB gene, bacteria were cleared from the blood.

The results presented in this thesis highlight the genetic and functional variability of pneumococcal strains within and between serotypes, and show that this variability influences the ability of individual strains to cause disease.

List of scientific papers

I. BROWALL, S., NORMAN, M., TANGROT, J., GALANIS, I., SJOSTROM, K., DAGERHAMN, J., HELLBERG, C., PATHAK, A., SPADAFINA, T., SANDGREN, A., BATTIG, P., FRANZEN, O., ANDERSSON, B., ORTQVIST, A., NORMARK, S., HENRIQUES-NORMARK, B. 2014. Intraclonal variations among Streptococcus pneumoniae isolates influence the likelihood of invasive disease in children. The Journal of infectious diseases, 209, 377-88.
https://doi.org/10.1093/infdis/jit481

II. SYK, A., NORMAN, M., FERNEBRO, J., GALLOTTA, M., FARMAND, S., SANDGREN, A., NORMARK, S., HENRIQUES-NORMARK, B. 2014. Emergence of hypervirulent mutants resistant to early clearance during systemic serotype 1 pneumococcal infection in mice and humans. The Journal of infectious diseases, 210, 4-13.
https://doi.org/10.1093/infdis/jiu038

III. NORMAN, M., GALLOTTA, M., NANNAPANENI, P., SENDER, V., PATHAK, A., BOOTSMA, H., BROWALL, S., JONCZYK, M., HASTE, L., HERMANS, P., ANDREW, P., HENRIQUES-NORMARK, B. Streptococcus pneumoniae strains with similar mortality show different patterns of disease progression in mice. [Manuscript]

IV. NORMAN, M., SYK, A., BROWALL, S., HENRIQUES-NORMARK, B. A bacteriophage contributes to virulence of Streptococcus pneumoniae serotype 1 during invasive disease. [Manuscript]