Regions of Diversity 8, 9 and 13 contribute to Streptococcus pneumoniae virulence

Abstract

Background: Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. Previously, using comparative genomic analyses, 13 regions of genomic plasticity have been identified in the S. pneumoniae genome. These "Regions of Diversity" (RDs) accounted for half the genomic variation observed amongst all pneumococci tested, moreover, were determined to encode a variety of putative virulence factors. To date, genes within 5 RDs have been unequivocally demonstrated to contribute to S. pneumoniae virulence. It is unknown if the remaining RDs also contribute to virulence.

Results: Using allelic exchange, we created S. pneumoniae mutants that were deficient in RD2, 5, 7, 8, 9, 12 and 13. Mutants deficient in RD8, 9 and 13 were attenuated in a mouse model of disease. RD8 is 40,358 nucleotides in length and encodes 37 genes. Using a panel of isogenic mutants, we determined that RD8b3 is the operon within RD8 that is responsible for virulence. Mice infected with mutants deficient in RD8, RD8b3, RD9 and RD13 had significantly less bacteria in the blood two days after intranasal challenge and improved survival over time versus mice infected with wild type. In all instances mutants colonized the nasopharynx at levels equivalent to wild type.

Conclusion: Genes within RD1, 3, 4, 6, and 10 have previously been shown to contribute to virulence. This study demonstrates that genes within RD8, 9 and 13 also contribute to virulence. The ability of mutants deficient in RD2, 5, 7, 8, 9, 12, and 13 to colonize the nasopharynx indicates that genes within these RDs are not required for asymptomatic carriage. Nonetheless, the observation that mutants deficient in RD8b3, 9 and 13 are attenuated indicates that genes within these loci are necessary for spread of the bacteria beyond the nasopharynx to normally sterile sites.

Figure 1

Schematic representation of RD8. RD8 is composed of two adjacent pathogenicity islands RD8a and RD8b [8]. Operons within RD8a and RD8b are shown.

Figure 2

Bacteria titers in the blood of mice challenged with RD deficient S. pneumoniae. Mice were challenged with 10⁷ cfu of TIGR4 and its isogenic RD mutants. Two days post-challenge, blood was collected from the tail vein of each mouse and the number of bacteria in the blood determined. Each circle represents the bacterial titer of an individual mouse. Bars indicate the median bacterial titer. Statistical analysis was performed using a Mann Whitney Rank Sum Test

Figure 3

Percent survival of mice following challenge with RD deficient S. pneumoniae. Kaplan-Meier plot illustrating survival of mice infected with wild type and RD deficient mutants. Cohorts of 10–11 mice were infected with wild type and the RD mutants. Survival was recorded over a 10 day period. Statistical analysis was performed using a Gehan-Breslow statistic analysis for survival.

Figure 4

Bacteria titers in the blood of mice challenged with isogenic mutants of RD8. Mice were challenged with 10⁷ cfu of TIGR4 and isogenic mutants deficient in operons within RD8. Two days post-challenge, blood was collected from the tail vein and the number of bacteria in the blood determined. Each circle represents the bacterial titer of an individual mouse. Bars indicate the median bacterial titer. Statistical analysis was performed using a Mann Whitney Rank Sum Test

Figure 5

Percent survival of mice following challenge with RD8 mutants. Kaplan-Meier plot illustrating survival of mice infected with wild type and RD8 operon deficient mutants. Cohorts of 11–12 mice were infected with wild type and the RD8 mutants. Survival was recorded over a 10 day period. Statistical analysis was performed using a Gehan-Breslow statistic analysis for survival.