Figure 4: In vivo phenotypes of SpnD39IIIA-F variant strains.

D39 wt or derivatives expressing only a locked SpnD39IIIA to SpnD39IIIF variant were used in these experiments. Carriage experiments were performed by intranasal inoculation of 5 × 10⁴ pneumococci into BALB/c mice, which are resistant to invasive infection. (a–c) The extent of carriage was evaluated by nasal lavage at day 1 (a), day 3 (b) and day 7 (c). (d,e) In the invasive disease model, susceptible CD1 mice received an intravenous challenge of 1 × 10⁵ pneumococci, and blood samples were taken at 4 and 30 h post challenge. Statistical significance of differing bacterial load in D39 wt or SpnD39III variant infected mice was calculated on log-transformed data using the unpaired (two-tailed) t-test. (f) Blood samples plated directly onto catalase agar plates were examined and the percentage of opaque colonies observed for each SpnD39III variant strain. (g,h) Allele quantification in D39 wt was performed on DNA extracted from the nasal lavages of the carriage experiment (see also Supplementary Table 5) or on DNA extracted from colonies grown from blood samples in the invasive disease experiment and SpnD39III variant percentages for each are represented respectively in g (inoculum black, 1 day grey, 3 days white, and 7 days striped) and h (inoculum black, 4 h grey and 30 h white). *P<0.05, **P<0.01, ***P<0.001.