Fig 1.
Model of glucose metabolism in S. pneumoniae.
Fig 2.
Deletion of spxB in TIGR4, but not in D39, demonstrates resistance to LL-37 killing.
The growth of cells in increasing concentration of LL-37 (0, 10, 21, and 42 μg/ mL) was measured. Strains include those in type 4 background—TIGR4 (A) and the spxB (B), lctO (C), and double spxB-lctO (D) mutants—and those in type 2 background—D39 (E) and the spxB (F), lctO (G), and double spxB-lctO (H) mutants.
Fig 3.
Capsule production is reduced in the TIGR4 spxB mutant.
Capsule production was measured in two methods. In the first, cell lysates from wild type and the spxB, lctO, and double spxB/lctO mutants in either TIGR4 (A) or D39 (B) backgrounds and the wild type and spxB mutant of the capsule swapped strain (TIGR4::D39) (C) were subjected to capsule blotting. In the second, immunoreactivity against capsule was determined using whole-cell bacterial ELISA for the TIGR4 (D), D39 (E), and TIGR4::D39 (F) strains. TIGR4R and D39 R6, acapsular variants, were included as a negative control (D, E). Capsule immunoreactivity was normalized to immunoreactivity to LytA, a cell surface protein, and then plotted as percentage of wild type. Mutants were compared to the wild type using unpaired parametric t-test; ** p = 0.01–0.001, *** p<0.001.
Fig 4.
Fluorescence against surface capsule is greatly diminished in the TIGR4 spxB mutant.
Capsule production was detected using fluorescence microscopy using primary antibodies against type 2 or 4 capsules with a fluorescent secondary (green) and DAPI stain to detect DNA (blue). Three representative images of TIGR4 (A) and the spxB (B), lctO (C), and double spxB lctO (D) mutants in the TIGR4 background and D39 (E) and the spxB (F), lctO (G), and double spxB lctO (H) mutants in the D39 background are shown.
Fig 5.
Capsule on the cell surface is undetectable in the TIGR4 spxB mutant.
Cell surface structures of TIGR4 (A) and the spxB (B), lctO (C), and double spxB lctO (D) mutants were imaged using transmission electron microscopy. Scale bars are 600 nm in the image and 200 nm in the inset.
Fig 6.
TIGR4 spxB mutant has reduced virulence in an IN mouse model.
BALB/c mice were infected IN with 1 x 107 cells. Bacterial presence in the blood in mice infected with TIGR4 strains was determined by detection of colony forming units (CFU/mL) at 24 hours (A) and 48 hours (B) post infection. In the same mice, bacterial carriage in the nasopharynx was determined at 24 hours (C) and 48 hours (D) post infection. Survival of mice was followed for 9 days (E). For each time-point of bacterial titers, mutant strains were compared to wild type using nonparametric Mann-Whitney t test; * p = 0.05–0.01, ** p = 0.01–0.001, *** p<0.001. Survival data were analyzed using the Mantel-Cox log rank test. p<0.0001 for TIGR4 spxB mutant compared to wild type; p = 0.0458 for TIGR4 lctO mutant compared to wild type; TIGR4 spxB lctO double mutant compared to wild type was non-significant.
Fig 7.
TIGR4 spxB mutant is not lethal in an IP mouse model.
BALB/c mice were infected IP with 1 x 103 cells and monitored for disease progression. Mice were infected with TIGR4 and the spxB, lctO, and double spxB-lctO mutants. Bacterial presence in the blood was determined at 24 hours (A) post infection. Survival of mice was followed for 8 days (B). For clarity purposes, the survival curves of TIGR4 and the lctO mutant was nudged. For blood titers, mutant strains were compared to wild type using nonparametric Mann-Whitney t test; ** p = 0.01–0.001. Survival data were analyzed using the Mantel-Cox log rank test. p = 0.0031 for TIGR4 spxB mutant compared to wild type; all other mutants compared to the wild type were non-significant.
Fig 8.
D39 spxB mutant maintains virulence in an IN mouse model.
BALB/c mice were infected IN with 1 x 107 cells. Bacterial presence in the blood in mice infected with D39 strains was determined at 24 hours (A) and 48 hours (B) post infection. In the same mice, bacterial carriage in the nasopharynx was determined at 24 hours (C) and 48 hours (D) post infection. Survival of mice was followed for 8 days (E). For each time-point of bacterial titers, mutant strains were compared to wild type using nonparametric Mann-Whitney t test; * p = 0.05–0.01. Survival data were analyzed using the Mantel-Cox log rank test. All mutants compared to the wild type were non-significant.
Fig 9.
Acetyl-CoA steady-state levels were reduced in the TIGR4 spxB mutant.
TIGR4 capsule consists of several sugars that are acetylated, unlike the capsule of D39. Capsule units are abbreviated: Pyr (pyruvate); Gal (galactose); ManNAc (N-acetyl-mannosamine); FucNAc (N-acetyl-fucosamine); GalNAc (N-acetyl-galactosamine); GlcA (glucuronic acid); Glc (glucose); Rha (rhamnose) (A). Acetyl-CoA levels were measured using mass spectrometry in wild type and the spxB, lctO, and double spxB-lctO mutants in the TIGR4 (B) and D39 (C) backgrounds. Acetyl-CoA values were normalized to total cellular protein and then plotted as pmol/ mg protein. Mutants were compared to the wild type using unpaired parametric t test; *** p<0.001.
Fig 10.
Disruption of pdhc reduces acetyl-CoA steady-state levels and abrogates capsule biosynthesis in TIGR4.
Acetyl-CoA levels were measured in the wild type and the pdhc mutant in TIGR4 background (A). Measurement of capsule polysaccharide produced by the pdhc mutant was determined by capsule blotting (B) and whole-cell bacterial ELISA (C). Acetyl-CoA values were normalized to total cellular protein and then plotted as pmol/ mg protein. The mutant was compared to the wild type using unpaired parametric t test; *** p< 0.001.
Fig 11.
Loss of capsule in spxB mutant is conserved and secondary mutations can reverse this phenotype.
Capsule production was measured using capsule blotting method. Strains used include wild type, spxB mutant, and spxB mutant revertant (rev) from four strains with serotype four capsule, including TIGR4, ABCA69, ABCB20, and ABCB54 (A). Capsule production was determined for spxB deletion in strains with capsules that contained different number of acetylated sugars, including 12F and the revertant and 45 (3 acetylated sugars), 19F and 35B (1 acetylated sugar), and 6B and 18C (0 acetylated sugars) (B).
Fig 12.
Deletion of spxB attenuates virulence and reduces steady-state acetyl-CoA in other pneumococcal strains with acetylated capsules.
BALB/c mice were infected IP with 5 x 104 cells. Bacterial presence in the blood in mice in strains with type 4 capsule was determined at 24 hours (A) and 48 hours (B) post infection and survival of mice was followed for 10 days (C). For strains with other capsule type—12F, 35B, 6B—bacterial presence in the blood in mice was determined at 24 hours (D) and 48 hours (E) post infection and survival of mice was followed for 10 days (F). Mice that did not survive to the 48 hour blood titer count are denoted by the symbol (x). Acetyl-CoA levels were measured in the wild type and spxB mutant in ABCB20, 12F, and 6B backgrounds (G). For each time-point of bacterial titers, mutant strains were compared to wild type using nonparametric Mann-Whitney t test; ** p = 0.01–0.001, *** p< 0.001. Survival data were analyzed using the Mantel-Cox log rank test. p< 0.0001 for ABCB20, ABCB54, and 6B spxB mutants compared to wild type; 12F and 35B spxB mutants compared to the wild type were non-significant. Acetyl-CoA values were normalized to total cellular protein and then plotted as pmol/ mg protein. The mutant was compared to the wild type using unpaired parametric t test; ** p = 0.01–0.001, *** p< 0.001.