Supporting Information

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Fig. 8. FACS analysis using RrgB antibodies of the invasive T4 strain (A), the clinical isolate of type 19F, ST162^19F (B), the deletion mutant of mgrA, T4D(mgrA) (C), and the nonpiliated mutant T4D(rrgA-srtD), lacking the rlrA islet (D), showing the proportion of piliated versus nonpiliated cells. Respectively, 84%, 90%, 99%, and 0% of the bacteria express pili.

Fig. 9. (A) Western blot analysis with RrgB antibodies, on a 4-12% gradient gel, detects HMW polymers in the mutant where the rlrA islet has been reintroduced into the deletion mutant T4D(rrgA-srtD). (B) Survival of mice after intranasal inoculation with 5 × 10⁶ (high dose) of the revertant where the rlrA islet has been reintroduced into the deletion mutant T4D(rrgA-srtD), as compared with T4D(rrgA-srtD) and T4. Virulence is restored to the same levels as for T4 having the rlrA islet. (C) The inflammatory response, after i.p. inoculation, is restored in the revertant where the rlrA islet has been reintroduced into the deletion mutant T4D(rrgA-srtD) (Mann-Whitney U test; *, P < 0.001). (D) Bacterial outgrowth in blood after i.p. challenge of mice with T4 and the revertant T4D(rrgA-srtD)Ñ(rrgA-srtD). (E) TNF response for individual mice correlated to the bacteremia levels after inoculation with T4 and the revertant T4D(rrgA-srtD)Ñ(rrgA-srtD).

Fig. 10. (A) Adherence of T4 and the mutant lacking rlrA (T4DrlrA) to monolayers of A549 lung epithelial cells. (B and C) Immunofluorescence microscopy of T4 (B), and T4D(rlrA) (C) adhering to A549 lung epithelial cells. Labeling of pneumococci with anti-capsular antibody (green) and visualization of epithelial F-actin with rhodamine (red) are shown. (D) Intranasal challenge of C57BL/6 mice with low/medium dose inoculation of T4 and T4D(rrgA-srtD), respectively, in separate experiments. Colonizations of individual mice are shown. The figure is a compilation of three different experiments.

Supporting Methods

FACS Analysis. Streptococcus pneumoniae [T4, ST162^19F, T4D (mgrA), and T4D(rrgA-srtD)] isolates were grown in THY liquid culture overnight at 37°C under 5% CO₂. Samples were diluted and allowed to grow to OD₆₀₀ = 0.250 (»1 × 10⁸ per ml). Bacterial cultures were centrifuged at 3,000 rpm and resuspended in 1× PBS. Fifteen microliters of bacterial suspension was added to 96-well plates. Five microliters of 20% normal rabbit serum was added to each well, along with primary antibodies (anti-RrgB, pre-immune anti-RrgB, and negative control Neisseria meningitidis anti-961) at 1:3,200. Samples were incubated on ice for 30 min, after which 150 ml of blocking buffer (1% BSA/PBS) was added to the wells. The 96-well plate was centrifuged at 2,500 rpm for 5 min at 4°C. A secondary anti-mouse antibody labeled with phycoerythrin (Jackson ImmunoResearch) was added at a final concentration of 1:100, and the mixture was incubated for 30 min on ice. Then 150 ml of blocking buffer was added and samples were centrifuged as above. Samples were resuspended in 200 ml of 1% paraformaldehyde/PBS and analyzed on the FACSCaliber (Becton Dickinson).

1. Sung, C. K., Li, H., Claverys, J. P. & Morrison, D. A. (2001) Appl. Environ. Microbiol. 67, 5190-5196.