Article Figures & Data
Figures
- Fig. 1.
Northern hybridization analysis of virulence-associated genes in CsrR− streptococcal strains. Bacterial RNAs from six strains were isolated in late exponential phase and hybridized with four different PCR-generated probes as indicated to the left of each panel. The source of each RNA sample is indicated above the respective lane. The fifth and sixth lanes in each panel contain RNA from derivatives of SBmuc5 and SBmuc7, respectively, into which acsrRS-bearing plasmid (pNLB2223) has been introduced. wt, wild type.
- Fig. 2.
Northern hybridization analysis of virulence-associated genes in CsrR− streptococcal strains. Samples from early-stationary-phase cultures of three strains were hybridized to probes specific for sagA, speB, andcpa. Samples included RNA from MGAS166, UMAA2392 (ΔcsrR csrS), UMAA2526 (ΔcsrR csrS ΔhasAB), and UMAA2392 with glucose added to a final concentration of 0.2% 2 h before RNA isolation.
- Fig. 3.
Hemolytic activities of streptococcal supernatants during 11 h of broth culture. (A) Absorbance of cultures from which aliquots were assayed. (B) Assays for SLS, performed in the presence of cholesterol to inhibit SLO. (C) Assays for SLO, performed in the presence of trypan blue to inhibit SLS. Note the different scales used in panels B and C. Data points for strain MGAS166 (wild type) are represented by the circles; those for strain UMAA2392 (ΔcsrR csrS) are represented by squares. White (open) and black (closed) data points indicate that assays were done in the absence or presence of horse serum, respectively. Assays were also performed without cholesterol and trypan blue and were within ±10% of the sum of the SLS and SLO activities (data not shown).
- Fig. 4.
Immunoblot analysis of streptococcal strains with a polyclonal antisera against SpeB. The three panels represent samples taken at different time intervals during growth in broth culture: mid-exponential (6 h of culture) (A), late exponential (12 h of culture) (B), and late stationary phase (48 h of culture) (C). Lanes were loaded with samples from the following strains: lane 1, MGAS166; lane 2, SBmuc5 (csrR::Tn916); lane 3, SBmuc7 (csrR::Tn916); lane 4, UMAA2392 (ΔcsrR csrS); lane 5, SBmuc5 (pNLB2223); and lane 6, SBmuc7 (pNLB2223). Lane M, molecular weight markers. The arrows indicate the mobility of 28-kDa proteins, corresponding to the molecular mass of fully processed SpeB. The slower-migrating bands that coexist in specimens containing specific SpeB bands are thought to represent unprocessed or complexed SpeB.
- Fig. 5.
Comparison of the size of necrotic lesions during the course of experiments II and IV (Table 3). In these experiments, necrotic lesions developed in all infected mice except in those inoculated with the wild-type strain, MGAS166 (no lesions), and in three of the mice inoculated with strain UMAA2526 (ΔcsrR csrS ΔhasAB). One of six mice inoculated with UMAA2526 in experiment II (A) and two of six mice in experiment IV (B) had no skin necrosis and are excluded from the analysis of lesion size in this figure. Lesions were measured, and the area was calculated 24, 48, and 72 h after inoculation. The infecting strains were SBmuc5 (solid black columns), SBmuc7 (white columns), UMAA2392 (cross-hatched columns), and UMAA2526 (shaded columns).
Tables
- Table 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Genotype or description Reference Strains MGAS166 M1 SpeA2, streptomycin resistant 25 SBmuc5 Tn916::csrR 5 SBmuc7 Tn916::csrR (reverse orientation of SBmuc5) 5 UMAA2392 MGAS166 ΔcsrR csrS (single base pair change in start codon [ATG→ACG]) This study NLB2226 MGAS166 (pNLB2223) 16 UMAA2497 MGAS166 ΔhasAB This study UMAA2526 UMAA2392 ΔhasAB This study UMAA2499 SBmuc5 (pASH2477) This study UMAA2353 SBmuc7 (pNLB2223) This study UMAA2561 UMAA2392 (pNLB2223) This study Plasmids pJRS233 emR, pGHOST4+oriV(Ts) (in streptococci) 28 pLZ12spec aad9, shuttle vector 17 pJRS525 aad9, shuttle vector with MCS and lacZ 23 pNLB2223 pLZ12spec carryingcsrR 16 pASH2477 pLZ12spec carrying csrRS This study pASH2233 pJRS233 carrying ΔcsrR This study pASH2468 pJRS233 carrying ΔhasAB This study - Table 2.
Levels of cell-associated uronic acids from GAS strain MGAS166 and derivative strains
Strain Phenotype Peak uronic acid level (μg/ml) MGAS166 Wild type 1.0 SBmuc5 Tn916::csrR 5.2 SBmuc7 Tn916::csrR 5.5 UMAA2392 ΔcsrR csrS 4.3 UMAA2497 ΔhasAB 1.1 UMAA2526 ΔcsrR csrS ΔhasAB 0.9 - Table 3.
Weight loss and lesion development in mice inoculated subcutaneously with GASa
Experiment (inoculum) Strain or description Mean 24-h weight gain ± SD No. with lesions/no. inoculatedb No. with necrosis/no. with lesionsb I (3 × 105) MGAS166 (wt) 1.3 ± 0.4 0/6 0/0 csrR::Tn916(muc5) 0.6 ± 0.5 1/6 0/1 ΔcsrR csrS −1.7 ± 1.6 4/6 3/4 ΔcsrR csrS ΔhasAB −0.1 ± 0.9 5/6 1/5 II (2 × 106) Controlc 0.6 ± 0.07 0/6 0/0 MGAS166 (wt) −0.4 ± 1.2 0/6 0/0 csrR::Tn916(muc5) −3.7 ± 0.8 6/6 6/6 ΔcsrR csrS −4.3 ± 0.6 6/6 6/6 ΔcsrR csrS ΔhasAB −3.4 ± 0.8 6/6 5/6 III (2 × 105) Controld 0.5 ± 0.6 0/8 0/0 MGAS166 (wt) 0.9 ± 0.4 0/6 0/0 csrR::Tn916(muc5) 0.2 ± 1.4 0/7 0/0 ΔcsrR csrS −2.1 ± 1.7 3/6 3/3 ΔcsrR csrS ΔhasAB 0.4 ± 1.3 1/6 0/1 IV (4 × 106) Controlc 1.2 ± 0.4 0/6 0/0 MGAS166 (wt) 1.1 ± 0.3 4/12 0/4 csrR::Tn916(muc5) −3.1 ± 0.6 6/6 6/6 csrR::Tn916(muc7) −3.3 ± 0.6 6/6 6/6 ΔcsrR csrS −3.7 ± 0.4 12/12 12/12 ΔcsrR csrS ΔhasAB −1.6 ± 0.9 6/6 4/6 ↵a All bacteria were grown in Todd-Hewitt broth at 37°C to mid-exponential phase (A600 = 0.6 to 0.7). Culture conditions were as follows: experiment I, shaken and aerated; experiment II, shaken and aerated; experiment III, static and capped; experiment IV, static and capped. wt, wild type.
↵b Counts taken at 72 h.
↵c Control mice were uninoculated mice from the same cohort.
↵d Control mice received subcutaneous injections of THY broth.
