Table 1.
Bacterial strains and plasmids used in this study.
Figure 1.
Influence of CcpA on GAS virulence factors is dependent on strain CovRS status.
TaqMan real-time QRT-PCR for (A) speB, (D) slo, (E) spyCEP, and (F) sagA was performed for cells grown to indicated growth phase (mid-exp = mid-exponential). From left to right bars represent: MGAS2221 (CovRS wild-type, green); 2221ΔccpA (blue); comp2221ΔccpA (red); MGAS5005 (CovS mutant, yellow); 5005ΔccpA (purple); and 5005compΔccpA (black). (B) Western immunoblot for SpeB in culture supernatants of indicated strains grown overnight in THY. The multiple bands observed represent zymogen (40 kb), intermediate, and mature (28 kb) SpeB forms reacting with anti-SpeB antibody [65]. (C) Casein hydrolysis of indicated strains as a measure of SpeB enzymatic activity. For all panels except (B) data graphed are mean +/− standard deviation of four biological replicates done on two separate occasions (i.e. eight samples). * indicates P<0.05 compared to CcpA inactivated strain as determined by ANOVA followed by Tukey's post-hoc test. speB, streptococcal pyrogenic exotoxin B; slo, streptolysin O; spyCEP, Streptococcus pyogenes cell envelope proteinase; sagA, streptolysin S.
Figure 2.
Recombinant GAS protein production and phosphorylation assays of recombinant GAS proteins.
(A) SDS-PAGE analysis of recombinant GAS proteins. (B) Recombinant HPr was incubated with HPrK/P, purified, and analyzed under native conditions as described in Materials and Methods. Phosphorylation of HPr results in faster gel migration under these conditions. (C) Recombinant CovR was phosphorylated using acetyl phosphate as described in Materials and Methods. Unphosphorylated and phosphorylated CovR were run as described for HPr. Phosphorylation of CovR results in dimerization and thus slower migration through the gel under these conditions.
Figure 3.
Recombinant CcpA-(HPr-Ser46-P) binds specifically to DNA from GAS genes encoding virulence factors.
Fluorescence polarization based assay of (A) CcpA-speB, (C) CcpA-nga/slo, and (D) CcpA-spyCEP cre interaction. (B) shows CcpA interaction with DNA from non-CcpA regulated gene (ftsX, i.e. negative control). Binding assays were done with (red circles) and without (blue circles) 10 µm HPr-Ser46-P. Representative fluorescence polarization-based binding isotherms are shown of experiments done on four occasions. For (A), (C), and (D) lines indicate non-linear fit of binding data as described in Materials and Methods. speB, streptococcal pyrogenic exotoxin B; ftsX, cell division protein; nga, NAD glycohydrolase; slo, streptolysin O; spyCEP, Streptococcus pyogenes cell envelope proteinase.
Figure 4.
Identification of significant overlap between the CcpA and CovR transcriptomes.
Differences in gene transcript levels measured by expression microarray analysis for select GAS virulence factor encoding genes (A) and genes involved in carbohydrate and amino acid utilization (B). Data graphed are mean +/− standard deviation for quadruplicate samples for strains as indicated in the insert box. All transcript levels are significantly different compared to strain MGAS2221 except for the emm gene which is shown for reference purposes. A significant difference was defined as at least two-fold difference in median gene transcript level and P<0.05 for the indicated isogenic mutant strain compared to the parental wild-type strain MGAS2221. speB, streptococcal pyrogenic exotoxin B; spyCEP, Streptococcus pyogenes cell envelope proteinase; endoS, endoglycosidase S; sagA, streptolysin S; slo, streptolysin O; emm, M protein; nanH, acetylneuraminate lyase; amyA, cyclomaltodextrin glucanotransferase; scrA, sucrose transport enzyme; arcA, arginine deiminase; hutI, imidazolonepropionase.
Table 2.
Selected genes/operons co-regulated by CcpA and CovR under laboratory conditions.
Figure 5.
Recombinant CovR and CcpA bind to DNA from promoter regions of the same genes.
(A-D) Representative fluorescence polarization-based isotherms of unphosphorylated (blue circles) and phosphorylated CovR (red circles, CovR-P) binding to 1 nM of fluorescein-labeled DNA. Millipolarization units (mP) are plotted against the CovR concentration. (A) Recombinant CovR interaction with DNA from the hasA promoter (positive control). (B) CovR interaction with DNA from promoter of the non-CovR regulated gene typA (i.e. negative control). For (B) note linear increase in MP values with increasing CovR concentration indicating low affinity protein-DNA interaction. (C) Recombinant CovR interacting with DNA from the amino acid utilization gene arcA. (D) CovR interaction with DNA from the carbohydrate utilization gene amyA. CcpA interaction with arcA cre (E) and amyA cre (F) is shown with (red circles) and without (blue circles) the presence of 10 µm HPr-Ser46-P. For all panels experiments were done on at least three occasions, and lines indicate fit of binding data as described in Materials and Methods. hasA, hyaluronan synthase; typA, GTP-binding protein; arcA, arginine deminase; amyA, cyclomaltodextrin glucanotransferase.
Figure 6.
CcpA and CovR contribute to GAS gene expression during growth in human saliva.
GAS strains (red bars = strain MGAS2221, yellow bars = strain 2221ΔccpA, and blue bars = strain 2221ΔcovR) were grown to late exponential phase in standard laboratory medium (THY) or human saliva and transcript level of indicated genes were assessed using TaqMan QRT-PCR. Quadruplicate replicates of each strains were assessed on two separate occasions for a total of eight replicates. * indicates P<0.05 for difference in mean transcript level of the indicated gene in human saliva compared with THY as determined by Student's t-test assuming unequal variances using Bonferroni's adjustment for multiple comparisons. speB, streptococcal pyrogenic exotoxin B; spyCEP, Streptococcus pyogenes cell envelope proteinase; sagA, streptolysin S; slo, streptolysin O; arcA, arginine deiminase; amyA, cyclomaltodextrin glucanotransferase.
Figure 7.
CcpA inactivation significantly decreases GAS virulence and affects GAS virulence gene expression during infection.
(A) 20 outbred CD-1 mice per GAS strain were infected intramuscularly with 2.5×107 CFU of indicated strains. Data graphed are survival over time with P values derived from Kaplan-Meier survival analysis. (B) Transcript levels of select GAS genes were determined in mouse muscle using QRT-PCR. From left to right bars represent 2221ΔccpA (red); 2221ΔcovR (yellow); and 2221ΔcovRΔccpA (blue). (C) Comparison of gene transcripts in strain MGAS2221 during infection compared with late exponential growth phase in THY. Comparison of gene transcripts between (D) strains MGAS2221 and 2221ΔccpA and (E) strains MGAS2221 and 2221ΔcovR during infection (blue bars) and during late exponential growth phase in THY (red bars). For (B-E) data graphed are mean +/− standard deviation of four biologic replicates analyzed in duplicate. (F) Bacterial density in muscle (lesion site) and spleen (disseminated infection site). Bars are as for panel A except that green bars represent strain MGAS2221. For all panels, * indicates P<0.05 for indicated comparison. spyCEP, Streptococcus pyogenes cell envelope proteinase; sagA, streptolysin S; slo, streptolysin O; endoS, endoglycosidase S; hasA, hyaluronan synthase; speB, streptococcal pyrogenic exotoxin B; emm, M protein; arcA, arginine deiminase; amyA, cyclomaltodextrin glucanotransferase; speG, streptococcal pyrogenic exotoxin G; mac-1, IgG degrading protease; scpA, streptococcal C5a peptidase; ska, streptokinase.
Table 3.
covRS mutations detected in GAS isolates from mice spleens following intramuscular inoculation with strain MGAS2221 and 2221ΔccpA.
Figure 8.
Model for how CcpA and CovR contribute to GAS gene expression profile.
Transport of environmental carbohydrates through the phosphotransferase system mediates HPrK/P kinase/phosphorylase activity thereby affecting the formation of HPr-Ser46-P and thus the interaction of CcpA with various cre sites. Similarly, CovS responds to environmental stimuli such as low Mg2+ concentration or the presence of the human cathelicidin LL-37 [51],[69] by altering the phosphorylation of status of CovR thereby modifying CovR-DNA interaction. The binding (or lack thereof) of CcpA and CovR to GAS DNA results in altered expression of genes encoding virulence factors, carbohydrate catabolism proteins, and amino acid catabolism proteins critical to the pathogenesis of GAS infection. Proteins that are freely secreted into the extracellular environment (e.g. SpeB) are shown without a surrounding ellipse. SpeB, streptococcal pyrogenic exotoxin B; SLS, streptolysin S; SpyCEP, Streptococcus pyogenes cell envelope proteinase; HasA, hyaluronan synthase; AmyA, cyclomaltodextrin glucanotransferase; ScrA, sucrose transport enzyme; ArcA; arginine deiminase; HutI, imidazolonepropionase.