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Molecular Pathogenesis

Effects of Oligopeptide Permease in Group A Streptococcal Infection

Chih-Hung Wang, Chia-Yu Lin, Yueh-Hsia Luo, Pei-Jane Tsai, Yee-Shin Lin, Ming T. Lin, Woei-Jer Chuang, Ching-Chuan Liu, Jiunn-Jong Wu
Chih-Hung Wang
Institute of Basic Medical Sciences
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Chia-Yu Lin
Departments of Microbiology and Immunology
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Yueh-Hsia Luo
Institute of Basic Medical Sciences
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Pei-Jane Tsai
Department of Laboratory Medicine and Biotechnology
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Yee-Shin Lin
Departments of Microbiology and Immunology
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Ming T. Lin
Institute of Medical Science, Medical College, Tzu Chi University, Hualien, Taiwan
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Woei-Jer Chuang
Biochemistry
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Ching-Chuan Liu
Pediatrics
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Jiunn-Jong Wu
Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng-Kung University, Tainan
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  • For correspondence: jjwu@mail.ncku.edu.tw
DOI: 10.1128/IAI.73.5.2881-2890.2005
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  • FIG. 1.
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    FIG. 1.

    (A) Map of the construction of the oppA isogenic mutant showing the BclI restriction sites in the oppA locus. The predicted hybridization fragments are also shown. (B) Southern blot assay of genomic DNA digested with BclI and extracted from GAS A-20 (lane 1), SW552 (lane 2), and SW553 (lane 3). A λ HindIII marker was used as a molecular size standard (lane M). The 1.97-kb probe used was specific for the oppA gene and λ DNA. (C) Transcription of oppA in the wild-type strain (A-20) at various times and in its oppA isogenic mutant (SW552). The results of RT-PCR were amplified with oppA Fwd-2 and Rev-2 primers. A 100-bp marker was used as a molecular size standard (lane M).

  • FIG. 2.
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    FIG. 2.

    Variations in hemolytic activities observed in time course assays with wild-type strain A-20 and opp isogenic mutant SW552. The growth curve for A-20 is also shown. (A) SLO activities of A-20 and SW552. (B) SLS activities of A-20 and SW552. The error bars indicate the standard deviations. The mean activities of SLO and SLS were calculated in three independent experiments.

  • FIG. 3.
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    FIG. 3.

    Effects of Opp on streptococcal virulence factor expression. (A) RT-PCR analysis of speF, sagA, speX, and slo. (B) Northern hybridization analysis of speF, sagA, speX, and slo. Streptococcal virulence gene transcription among wild-type strain A-20, opp isogenic mutant SW552, opp revertant SW553, and opp complementation strain SW563 is shown.

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    FIG. 4.

    RT-PCR analysis of the expression of streptococcal regulators and virulence factors during various growth periods. RT-PCR-amplified products of sagB (A), pel (B), slo (C), rofA (D), and dppA (E) were made from cell harvested from strains A-20 and SW552 at 5, 7, 9, and 11 h. Time zero is defined as the time when the bacterial suspension was transferred to fresh medium. The transcript of 16S rRNA was used as an internal control. The extent of expression was normalized to the ratio of virulence gene RNA and 16S rRNA. The data represent the means and standard deviations obtained from at least three independent experiments.

  • FIG. 5.
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    FIG. 5.

    Survival of GAS-infected mice after inoculation in air pouches with 109 CFU of wild-type strain A-20 (n = 9), speB mutant SW507 (n = 9), opp mutant SW552 (n = 12), and opp complementation strain SW563 (n = 10). These mice were injected subcutaneously with about 1 ml of air to form an air pouch, and then a bacterial suspension (0.1 ml) was inoculated into the air pouch. The mortality rates were monitored every day during the experimental period. Bacterial numbers were determined by measuring the OD600 with a spectrophotometer.

Tables

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  • TABLE 1.

    Specific PCR primer sets used in RT-PCR analysis

    GenePrimeraSequence (5′→3′)Start siteReference or sourceb
    speBFwdGGATCCATATGGATCAAACTTTGCTCGTAACG8112
    RevGAATTCGGATCCTAAGGTTTGATGCCTACAACAG1175
    speFFwdATGGCGCAAGCAAGTACCTA97731
    RevTTTGAGTAGGTGTACCTTAT1624
    speXFwdTTTCTCGTCCTGTGTTTGGA12210
    RevTTGTGATATTAAAAATTTTCTCGCC574
    speGFwdACCCCATGCGATTATGAAAA47978
    RevGGGAGACCAAAAACATCGAC4966
    speJFwdTCTTTCATGGGTACGGAAGTG41This study
    RevAGCTCTCGACCTCAGAATCAA238
    sagAFwdCTCGCGTTCTTATCAGTTAC1402
    RevACCTGGCGTATAACTTCC339
    sagBFwdTGTCCGCCAATAACTGTTGA727This study
    RevCACCGTATTCCGCAAAATCT1365
    sloFwdACTCTGTCACTGATAGGACC698This study
    RevGAGCTGCTTCAATCTGTG998
    csrSFwdTGGGTTTTCCATGACACAAA140023
    RevAGCGCCGCGTAGTAATTAAG1594
    csrRFwdTGCAACATGAGGGGTATGAA22023
    RevTAAACCTGCAACCACATCCA531
    emm1FwdAACGGCTTCAGTAGCGGTAG108211
    RevCTGCAACTTCCATTGCATTC1281
    sdaD (DNase)FwdCACAACAGCCAGGGAATTTT92537
    RevGATGGTCTTGGTCCTCCTTG1105
    16S rRNAFwdGCAGCAGTAGGGAATCTTCG330This study
    RevCGCTCGGGACCTACGTATTA530
    oppAFwd-1ATGAAGAAAAGTAAATGGTT138236
    Fwd-2GATCCACGGACCTACCTTGA2901
    Rev-1TTATTTTTCAACGTGATCAG3352
    Rev-2CAGCTGCCACAACATCCTTA3003
    dacAFwd-1AGGCGATGACCCACTAGGGA101536
    Fwd-2TATTAGCTCCTAAAAATG559
    dppAFwdGAAAACTGCAGTGACTGAGGCGCATTCGACA124735
    RevGGCCGGAATTCTCAGAAGATGTCATTGCTT2189
    rofAFwdATCGCTCTGCTATATAGTAAG520This study
    RevACCGTCTCAGTGCTATCAA2031
    pelFwdAGGAGGTAAACCTTATGTT24
    RevGCTAAATAGATTATTTACCTG
    oppFRevTTACAATTCTTTTTGATATT784036
    • ↵a Fwd, forward; Rev, reverse.

    • ↵b Primers for which the source is listed as this study were predicted according to Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) calculations. The accession numbers for speJ, sagB, slo, 16S rRNA, and rofA are AF321000, AF067649, A28468, AB023575, and U01312, respectively, in the National Center for Biotechnology Information nucleotide database.

  • TABLE 2.

    SLO activities in oppA-positive and oppA-negative mutant strains of GAS in various atmospheres

    AtmosphereMean ± SEM % of SLO hemolysis ina:
    A-20SW552SW553
    20% O2, 0.5% CO220.00 ± 1.1b44.4 ± 1.124.2 ± 4.3
    20% O2, 5% CO225.20 ± 3.8b43.5 ± 1.425.2 ± 1.3
    5% O2, 10% CO210.90 ± 0.5b17.7 ± 1.412.2 ± 1.8
    • ↵a The hemolytic activity of SLO was measured as described in the text. The results are from three experiments. Student's t test was used for statistical analysis.

    • ↵b The P value in a comparison with SW552 was <0.05.

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Effects of Oligopeptide Permease in Group A Streptococcal Infection
Chih-Hung Wang, Chia-Yu Lin, Yueh-Hsia Luo, Pei-Jane Tsai, Yee-Shin Lin, Ming T. Lin, Woei-Jer Chuang, Ching-Chuan Liu, Jiunn-Jong Wu
Infection and Immunity Apr 2005, 73 (5) 2881-2890; DOI: 10.1128/IAI.73.5.2881-2890.2005

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Effects of Oligopeptide Permease in Group A Streptococcal Infection
Chih-Hung Wang, Chia-Yu Lin, Yueh-Hsia Luo, Pei-Jane Tsai, Yee-Shin Lin, Ming T. Lin, Woei-Jer Chuang, Ching-Chuan Liu, Jiunn-Jong Wu
Infection and Immunity Apr 2005, 73 (5) 2881-2890; DOI: 10.1128/IAI.73.5.2881-2890.2005
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