Capacity To Utilize Raffinose Dictates Pneumococcal Disease Phenotype

Genetic loci encoding raffinose uptake and utilization in S. pneumoniae. The numbers below each gene refer to the locus tags in the serotype 14 ST15 947 genome. The locations of SNPs in serotype 14 ST15 and serotype 3 ST180 isolates are indicated with asterisks; horizontal arrows show the locations of promoters.

Expression of raffinose pathway genes by serotype 14 and 3 blood and ear isolates. The indicated strains were grown in CDM+Glc to an OD₆₀₀ of 0.2, washed and resuspended in CDM+Raf, and then incubated at 37°C for a further 30 min. RNA was then extracted, and levels of aga, rafG, and rafK mRNA were analyzed by qRT-PCR using 16S rRNA as an internal control (see Materials and Methods). The data presented are the means ± SD from three independent experiments. *, P < 0.05, **, P < 0.01, and ****, P < 0.0001, by unpaired t test.

Growth phenotype and raffinose pathway gene expression in serotype 23F ST81 blood and ear isolates. (A) Growth of blood isolate 5076 and ear isolate 9725241 in CDM+Glc or CDM+Raf was monitored by OD₆₀₀ for 12 h. Data are mean OD₆₀₀ ± SD from triplicate assays. (B) The indicated strains were grown in CDM+Glc to an OD₆₀₀ of 0.2, washed and resuspended in CDM+Raf, and then incubated at 37°C for a further 30 min. RNA was then extracted, and levels of aga, rafG, and rafK mRNA were analyzed by qRT-PCR using 16S rRNA as an internal control. The data presented are the means ± SD from three independent experiments. ***, P < 0.001 by unpaired t test.

Growth phenotype and raffinose operon gene expression in rafR exchange mutants. (A) S. pneumoniae strains 4559, 947, 4559^947rafR, and 947^4559rafR were grown in CDM+Glc or CDM+Raf, and OD₆₀₀ was monitored for 12 h. Data are mean OD₆₀₀ ± SD from triplicate assays. (B) The indicated strains were grown in CDM+Glc to an OD₆₀₀ of 0.2, washed and resuspended in CDM+Raf, and then incubated at 37°C for a further 30 min. RNA was then extracted, and levels of aga, rafG, and rafK mRNA were analyzed by qRT-PCR. Data are the means ± SD from three independent experiments. *, P < 0.05, ***, P < 0.001, and ****, P < 0.0001, by unpaired t test.

Virulence phenotype of rafR exchange mutants. Groups of 16 mice were infected intranasally with 10⁸ CFU of the indicated strain. At 24 h, all mice from each group were euthanized and numbers of pneumococci in the indicated tissues/sites were quantitated (see Materials and Methods). Viable counts (total CFU per tissue) are shown for each mouse at each site; horizontal bars indicate the geometric mean (GM) CFU for each group; the broken line indicates the threshold for detection. Differences in GM bacterial loads between groups are indicated by asterisks: *, P < 0.05, **, P < 0.01, and ****, P < 0.0001, by unpaired t test.