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MOLECULAR AND CELLULAR PATHOGENESIS

A Two-Component Regulatory System, CsrR-CsrS, Represses Expression of Three Streptococcus pyogenesVirulence Factors, Hyaluronic Acid Capsule, Streptolysin S, and Pyrogenic Exotoxin B

Andrew Heath, Victor J. DiRita, Neil L. Barg, N. Cary Engleberg
Andrew Heath
Departments of Internal Medicine and
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Victor J. DiRita
Microbiology and Immunology, and the
Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109
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Neil L. Barg
Departments of Internal Medicine and
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N. Cary Engleberg
Departments of Internal Medicine and
Microbiology and Immunology, and the
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DOI: 10.1128/IAI.67.10.5298-5305.1999
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  • Fig. 1.
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    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.

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    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.

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    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).

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    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.

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    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

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  • Table 1.

    Bacterial strains and plasmids used in this study

    Strain or plasmidGenotype or descriptionReference
    Strains
     MGAS166M1 SpeA2, streptomycin resistant25
     SBmuc5Tn916::csrR5
     SBmuc7Tn916::csrR (reverse orientation of SBmuc5)5
     UMAA2392MGAS166 ΔcsrR csrS (single base pair change in start codon [ATG→ACG])This study
     NLB2226MGAS166 (pNLB2223)16
     UMAA2497MGAS166 ΔhasABThis study
     UMAA2526UMAA2392 ΔhasABThis study
     UMAA2499SBmuc5 (pASH2477)This study
     UMAA2353SBmuc7 (pNLB2223)This study
     UMAA2561UMAA2392 (pNLB2223)This study
    Plasmids
     pJRS233emR, pGHOST4+oriV(Ts) (in streptococci)28
     pLZ12specaad9, shuttle vector17
     pJRS525aad9, shuttle vector with MCS and lacZ23
     pNLB2223pLZ12spec carryingcsrR16
     pASH2477pLZ12spec carrying csrRSThis study
     pASH2233pJRS233 carrying ΔcsrRThis study
     pASH2468pJRS233 carrying ΔhasABThis study
  • Table 2.

    Levels of cell-associated uronic acids from GAS strain MGAS166 and derivative strains

    StrainPhenotypePeak uronic acid level (μg/ml)
    MGAS166Wild type1.0
    SBmuc5Tn916::csrR5.2
    SBmuc7Tn916::csrR5.5
    UMAA2392ΔcsrR csrS4.3
    UMAA2497ΔhasAB1.1
    UMAA2526ΔcsrR csrS ΔhasAB0.9
  • Table 3.

    Weight loss and lesion development in mice inoculated subcutaneously with GASa

    Experiment (inoculum)Strain or descriptionMean 24-h weight gain ± SDNo. with lesions/no. inoculatedbNo. with necrosis/no. with lesionsb
    I (3 × 105)MGAS166 (wt)1.3 ± 0.40/60/0
    csrR::Tn916(muc5)0.6 ± 0.51/60/1
    ΔcsrR csrS−1.7 ± 1.64/63/4
    ΔcsrR csrS ΔhasAB−0.1 ± 0.95/61/5
    II (2 × 106)Controlc0.6 ± 0.070/60/0
    MGAS166 (wt)−0.4 ± 1.20/60/0
    csrR::Tn916(muc5)−3.7 ± 0.86/66/6
    ΔcsrR csrS−4.3 ± 0.66/66/6
    ΔcsrR csrS ΔhasAB−3.4 ± 0.86/65/6
    III (2 × 105)Controld0.5 ± 0.60/80/0
    MGAS166 (wt)0.9 ± 0.40/60/0
    csrR::Tn916(muc5)0.2 ± 1.40/70/0
    ΔcsrR csrS−2.1 ± 1.73/63/3
    ΔcsrR csrS ΔhasAB0.4 ± 1.31/60/1
    IV (4 × 106)Controlc1.2 ± 0.40/60/0
    MGAS166 (wt)1.1 ± 0.34/120/4
    csrR::Tn916(muc5)−3.1 ± 0.66/66/6
    csrR::Tn916(muc7)−3.3 ± 0.66/66/6
    ΔcsrR csrS−3.7 ± 0.412/1212/12
    ΔcsrR csrS ΔhasAB−1.6 ± 0.96/64/6
    • ↵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.

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A Two-Component Regulatory System, CsrR-CsrS, Represses Expression of Three Streptococcus pyogenesVirulence Factors, Hyaluronic Acid Capsule, Streptolysin S, and Pyrogenic Exotoxin B
Andrew Heath, Victor J. DiRita, Neil L. Barg, N. Cary Engleberg
Infection and Immunity Oct 1999, 67 (10) 5298-5305; DOI: 10.1128/IAI.67.10.5298-5305.1999

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A Two-Component Regulatory System, CsrR-CsrS, Represses Expression of Three Streptococcus pyogenesVirulence Factors, Hyaluronic Acid Capsule, Streptolysin S, and Pyrogenic Exotoxin B
Andrew Heath, Victor J. DiRita, Neil L. Barg, N. Cary Engleberg
Infection and Immunity Oct 1999, 67 (10) 5298-5305; DOI: 10.1128/IAI.67.10.5298-5305.1999
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KEYWORDS

Bacterial Capsules
Bacterial Proteins
Cysteine Endopeptidases
Hyaluronic Acid
Operon
Streptococcus pyogenes
Streptolysins

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