Figure 1.
Differences in surface expression of pneumococcal ligands is dependent on serotype.
A) Immunoblot analysis comparing relative protein levels in whole cell lysates of TIGR4, an unencapsulated derivate (T4R), and the panel of capsular switch mutants. Whole cell lysates (10 µg) were separated by 12%SDS-PAGE and transferred to nitrocellulose or directly blotted onto membranes and probed with antisera to choline binding protein (CbpA), pneumococcal surface protein A (PspA), or the pneumococcal serine-rich repeat protein (PsrP). B) Capture of bacteria to immobilized antibodies to CbpA (black bars) and PsrP (white bars). TIGR4 and the panel of isogenic mutants were incubated in wells pre-coated with serum to CbpA or PsrP and surface expression was indicated by the amount of bacteria captured to immobilized antibody. Values are presented as a percentage of capture of the TIGR4 strain. Statistical analysis was performed using a two-tailed Student's t-test versus TIGR4. Single asterisk indicates P<0.05, double asterisks indicates P<0.001.
Figure 2.
Effect of serotype and surface accessibility of pneumococcal ligands.
Flow cytometry histograms for pneumococcal surface ligands CbpA, PspA, and PsrP in A) TIGR4 and its unencapsulated derivative (T4R) and B) the isogenic capsular switch strains +4, +6A, +7F, +23F. Histograms are representative of three independent experiments using bacteria collected from overnight plates (similar results were obtained using planktonic pneumococci). Surface accessibility of CbpA, PspA, and PsrP as measured by mean fluorescence intensity (MFI; black bars) and %PE+ positively labeled cells (white bars). Results from three independent experiments are shown. Statistical analysis was performed using a two-tailed Student's t-test versus T4. Single cross indicates P<0.05, double cross indicates P<0.001.
Figure 3.
Effect of serotype on bacterial adhesion.
In vitro adhesion assays measuring the ability of TIGR4 or the isogenic capsular switch mutants to adhere to A) human lung alveolar (A549) and B) nasopharyngeal (Detroit) cell lines. Adhesion is expressed as a percentage relative to TIGR4 adhesion. Statistical analysis was performed using a two-tailed Student's t-test versus TIGR4. Single asterisk indicates P<0.05, double asterisks indicates P<0.001.
Figure 4.
Genetic switch of serotype alters nasopharyngeal colonization and virulence in mice.
Bacterial titers were measured for individual 5-week old female BALB/cJ mice intranasally infected with 106 CFU of the parental TIGR4 WT (n = 8) or the isogenic capsular switch strains +6A, +7F, +23F, and +4 (n = 6). A) Nasal lavages and B) blood were collected at 24, 72, and 120 h post-infection. Horizontal bars represent the median value. Statistical analysis was performed using One-Way ANOVA in comparison to both the TIGR4 strain (*) and the internal +4 control strain (+). Asterisk and the cross indicate a significance value of P<0.05.
Figure 5.
Serotype affects virulence in the lungs.
Bacterial titers were measured for individual 5-week old female BALB/cJ mice infected via A) intratracheal (105 CFU) or B) intraperitoneal (104 CFU) routes with TIGR4 WT (n = 6) or the isogenic capsular switch strains +6A, +7F, +23F, and +4 (n = 6). For intratracheal challenge samples were collected at 48 h post-infection, and for intraperitoneal challenge samples were collected at 24 and 48 hours. Horizontal bars represent the median value. Statistical analysis was performed using One-Way ANOVA in comparison to both the TIGR4 strain (*) and the internal +4 control strain (+). Asterisk and the cross indicate a significance value of P<0.05.
Figure 6.
Clearance of FAMSE-labeled capsule switch strains from the lungs and uptake by alveolar macrophages 4 hours-post inoculation.
A) Alveolar macrophage phagocytosis of CPS switch strains as determined by FACS. Data presented as the median (IQR) fluorescence index (percentage of fluorescent macrophages multiplied by the geo mean MFI) for BALF macrophages (n = 5). B) Median bacterial titers in bronchoalveolar lavage fluid (BALF) (n = 5). For panels A and B samples were collected 4 hours after intranasal challenge with 5×106 CFU FAMSE-labeled CFU, and the P values given above the respective box and whisker plot for individual strains are for comparison to the +4 strain. A Dunn's Multiple Comparison test was used for statistical analyses.