Fig 1.
Deletion of S. pyogenes sortase results in aberrant cellular morphology.
Wild type S. pyogenes D471, sortase mutant AR01, complemented AR01+pAR107, and a variant of AR01 that was passaged 6 times in TH+Y, were diluted from overnight cultures 1:100 into fresh media (containing spectinomycin for AR01+pAR107), grown to log phase, and fixed. The cells were processed for transmission and scanning electron microscopy as described in the Material and Methods section. Scale bars represent 500 nm.
Fig 2.
Missorted surface proteins accumulate in the sortase mutant AR01.
A. Wild type S. pyogenes D471, sortase mutant AR01, complemented AR01+pAR107, and a passaged AR01 variant, were diluted from overnight cultures 1:100 into fresh media (containing spectinomycin for AR01+pAR107), grown to log phase, fixed, attached to glass cover slides, and permeabilized with PlyC. The cells were blocked and incubated with pentaglycine-biotin (5G-biotin) in the presence or absence of purified S. aureus sortase A. The slides were washed, and pentaglycine-biotins molecules (attached to LPXTG motifs by sortase) were detected using FITC-streptavidin. DAPI was used to visualize DNA. The scale bar represents 1 μm. Note that expression of sortase from pAR107 is lower than the wild type, explaining the presence of intact LPXTG motifs. B. The cell wall fraction of log-phase D471 cells (solubilized with PlyC in PBS 30% raffinose) was separated by SDS-PAGE. Duplicate gels were stained with GelCode blue or processed for Western blot using the 10B6 monoclonal antibody. The most prominent bands on the stained gel correspond to M protein.
Fig 3.
Passage of the sortase mutant AR01 in TH+Y results in loss of M protein expression.
A Wild type S. pyogenes D471, sortase mutant AR01, complemented AR01+pAR107, and 10 separate variants of AR01 that were passaged 6 times in TH+Y, were diluted from an overnight culture 1:100 into fresh media (containing spectinomycin for AR01+pAR107), grown to log phase, fractionated into supernatant, wall (solubilized with PlyC in PBS 30% raffinose), and spheroplast pellet, and examined by Western blot. The monoclonal antibody 10B6 was used to detect M protein, specific serum was used to detect SfbI, and the monoclonal antibody 3A1 was used to detect cytoplasmic glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as loading control. B Cells grown in a similar manner were fixed, and processed for fluorescence microscopy as described in the Materials and Methods section. Specific antibodies were used to label M protein (red) and SfbI (green). The cell wall was stained with WGA marina blue (blue). Images were obtained using deconvolution immunofluorescence and Nomarski microscopy. Deconvolution images are presented as maximum intensity projections, composed of all the Z-sections. Additional images are presented in S4 Fig.
Fig 4.
The S. pyogenes sortase mutant is under selective pressure leading to the loss of M protein expression.
A Wild type D471, sortase mutant AR01 (original stock), and complemented AR01+pAR107, were sonicated to separate the streptococcal chains into single cells, and subsequently plated. Single colonies were picked and grown in TH+Y (containing spectinomycin for AR01+pAR107) at 37°C overnight. Each day, a seed culture was transferred to a new tube for overnight growth, and a sample of the original culture was separated into supernatant and cell pellet, and stored at -80°C until use; the cultures were passaged in this manner six times. The cell pellets were lysed with PlyC. M protein was quantified by capture ELISA as described in the Materials and Methods section. The results represent 13 repeats for D471, 24 repeats for AR01, and 11 repeats for AR01+pAR107. B The relative amount of SfbI in AR01 supernatant and cell lysate was similarly determined by capture ELISA; the results represent 15 repeats. See S6 Fig for sfbI-negative control.
Fig 5.
Deletion of sortase in a strain lacking M protein does not lead to morphological aberrations.
A Wild type D471, D471-derived sortase mutants (original AR01, and new AR01.1-AR01.4), M-negative JRS75, and JRS75-derived sortase mutants (AR03.1-AR03.4), were each diluted from an overnight culture 1:100 into fresh TH+Y and grown to log phase. Cultures were fractionated into supernatant, wall (solubilized with PlyC in PBS 30% raffinose), and spheroplast pellet, and examined by Western blot. The monoclonal antibody 10B6 was used to detect M protein, and specific serum was used to detect SfbI.B Cells grown in a similar manner were fixed and examined by scanning electron microscopy as described in the Materials and Methods section; the scale bar represents 1 μm. Note that while one of the D471-derived sortase mutants (AR01.3) did not display morphological aberrations, this strain had lost the expression of M protein early, as observed by Western blot.
Fig 6.
The sortase mutant, but not the passaged strain, displays increased membrane permeability.
Wild type D471, sortase mutant AR01 (original stock), complemented AR01+pAR107, and a low-M passaged variant of AR01, were diluted 1:50 from an overnight culture and grown to OD600 0.5. The cells were incubated with 5 mM SYTOX green for 30 min at room temperature, washed with PBS, and immediately imaged. The scale bar represents 2 μm. Additional images are presented in S8 Fig.
Fig 7.
The sortase mutant is highly sensitive to LL-37.
A Wild type D471, original sortase mutant AR01, sortase complemented AR01+pAR107, and passaged AR01 (day 6), were grown to OD600 0.5, diluted 1:105 in TH+Y (containing spectinomycin for AR01+pAR107) and mixed with LL-37, scrambled peptide, or TH+Y alone. The strains were incubated stationary in a 96-well plate for 16 h at 37°C, and the final OD600 was measured. The final OD600 value is presented as a fraction of the value obtained in the absence of peptides (defined as 100%). Experiments were repeated 3 times in triplicates, and a representative experiment is shown; error bars represent standard deviation. P-values for samples showing significant growth inhibition were calculated using t-test. B Cultures prepared in a similar manner were rotated in microfuge tubes for 3 h at 37°C, and then serially diluted and plated for CFU quantification. The CFU ratio treated/untreated is presented. Experiments were repeated 3 times in duplicates, and the results from all 3 experiments are shown; error bars represent standard deviation. P-values were calculated using t-test.
Fig 8.
pyogenes sortase mutant is susceptible to phagocytosis in human blood, and grows poorly in plasma.
S. Wild type D471, original sortase mutant AR01, sortase-complemented AR01+pAR107, and passaged AR01 (day 6), were grown to OD600 0.2, diluted 1:105, and incubated in the following conditions: 1) rotated in blood, 2) stationary in blood (phagocytes sink to the bottom), 3) rotated in blood + 20 μM cytochalasin B (inhibits phagocytosis), 4) rotated in plasma, 5) rotated in TH+Y. Blood and plasma were from a healthy donor who tested negative for antibodies specific to M protein serotype 6. Following 3 h incubation at 37°C, samples were serially diluted 10-fold, and plated on TH+Y for CFU quantification; results are presented as CFUfinal/CFUstart. Experiments were repeated 3 times in duplicate, and the results from all 3 experiments are shown; error bars represent SEM. P values compared to wild type D471 were calculated using t-test; “ns” denotes no statistical significance. # symbol denotes that one or more data points in the group were below detection level.
Fig 9.
Expression of M protein with altered LPXTG motif in wild type D471 reduces fitness in the host environment.
A D471 containing plasmids pAR161 (empty), pAR186 (LPSTGE), pAR184 (EGTSPL), or pAR185 (TEPGSL), was diluted from an overnight culture 1:100 into fresh TH+Y containing spectinomycin, grown to log phase, and fractionated to supernatant, cell wall, and spheroplast fraction pellet fractions. Fractions were analyzed by Western blot, and M protein was detected using the monoclonal 10B6. B The four strains were grown to OD600 0.2, diluted 1:105, and incubated in the following conditions: 1) rotated in blood, 2) stationary in blood (phagocytes sink to the bottom), 3) rotated in blood + 20 μM cytochalasin B (inhibits phagocytosis), 4) rotated in plasma, 5) rotated in TH+Y. Blood and plasma were from a healthy donor who tested negative for antibodies specific to M protein serotype 6. Following 3 h incubation at 37°C, samples were serially diluted 10-fold, and plated on TH+Y for CFU quantification; results are presented as CFUfinal/CFUstart. Experiments were repeated 3 times in duplicate, and the results from all 3 experiments are shown; error bars represent SEM. P values compared to wild type D471 were calculated using t-test; “ns” denotes no statistical significance.