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Molecular Pathogenesis

Inorganic Phosphate Induces Spore Morphogenesis and Enterotoxin Production in the Intestinal Pathogen Clostridium perfringens

Valeria A. Philippe, Marcelo B. Méndez, I-Hsiu Huang, Lelia M. Orsaria, Mahfuzur R. Sarker, Roberto R. Grau
Valeria A. Philippe
1Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Rosario, Argentina
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Marcelo B. Méndez
1Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Rosario, Argentina
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I-Hsiu Huang
2Department of Microbiology, College of Science
3Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon
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Lelia M. Orsaria
1Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Rosario, Argentina
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Mahfuzur R. Sarker
2Department of Microbiology, College of Science
3Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon
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Roberto R. Grau
1Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Rosario, Argentina
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  • For correspondence: robertograu@fulbrightweb.org
DOI: 10.1128/IAI.02090-05
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  • FIG. 1.
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    FIG. 1.

    Western blot analysis of CPE production under Pi-regulated conditions. Cultures of the wild-type strain SM101 were grown in DSMM medium with or without the addition of Pi (35 mM). At the indicated times, samples were removed to be analyzed for enterotoxin production using specific anti-CPE antibodies (11, 20). Control lane (data not shown) containing purified CPE confirmed the identity of the detected band. Results from a representative experiment are shown.

  • FIG. 2.
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    FIG. 2.

    Efficiency of spore formation under Pi-buffered or Tris-buffered conditions. Cultures of the wild-type strain SM101 were grown in DSMM supplemented with different amounts of 1 M Tris-HCl, pH 8.0, or 1 M Na2HPO4 for 20 h at 37°C. The final pH after growth and the final cellular yield (circles) and spore efficiency (triangles) under each growth condition are indicated. Open symbols, Tris-buffered cultures; filled symbols, Pi-buffered cultures. The results shown are the average of five independent experiments; bars indicate standard errors. Similar effects of Pi on spore efficiency were obtained with other enterotoxigenic C. perfringens human isolates other than SM101 (data not shown). Essentially the same results as the ones observed in DSMM-Tris were obtained when the Tris buffer was replaced by MOPS buffer (DSMM-MOPS; data not shown).

  • FIG. 3.
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    FIG. 3.

    cpe expression and CPE production under different growth conditions. (A) Wild-type cultures of a derivative SM101 strain carrying a cpe-gusA plasmid were grown in DSMM with or without the addition of Pi (35 mM) and/or glucose (1%). Samples were removed at the indicated times and analyzed for β-glucuronidase activity as indicated (30). ○, DSMM plus Pi; ▵, DSMM alone; ⋄, DSMM plus Pi and glucose; and □, DSMM plus glucose. (B) Cultures carrying the cpe-gusA reporter were developed in three different media able to support the growth of C. perfringens: DSMM, TY, and FT medium with or without the addition of Pi (35 mM; see text for details). The bars indicate the level of β-glucuronidase activity accumulated by 1 h after the end of the exponential phase of growth. (C) Western blotting experiment using anti-CPE antibodies (20) of C. perfringens cultures grown in the indicated medium with or without supplementation with inorganic phosphate. The cellular pellet from the culture grown in DSMM with Pi was diluted 100-fold, while all the other pellets were undiluted. Data from representative experiments are shown.

  • FIG. 4.
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    FIG. 4.

    Cell phenotypes of C. perfringens grown in the presence or absence of Pi signaling. (A to D) Phase-contrast photomicrographs of Spo0A-proficient (SM101) and Spo0A-deficient cells (IH101) grown for 5 h in DSMM with or without added Pi. Wild-type cells (SM101) grown in the presence of added Pi displayed polar prespores (A), while the non-Pi-supplemented culture (B) showed cells with a cellular phenotype resembling spo0A mutants (11) blocked at stage zero of sporulation, regardless of whether inorganic phosphate was added or not (C and D). After the overnight growth of the wild-type cultures, no prespores were observed in the non-Pi-supplemented culture, while in the Pi-supplemented culture a great proportion of mature and free spores (more than 60%) were observed (data not shown). (E to H) Fluorescent photomicrographs with DAPI (specific for DNA staining) and FM 4-64 (specific for membrane lipid staining) of wild-type cells (SM101) grown for 5 h in DSMM with or without supplementation with Pi.

  • FIG. 5.
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    FIG. 5.

    Phosphate regulates the onset of sporulation in C. perfringens. Northern blot analysis of spo0A levels under Pi and glucose regulatory growth conditions. Total RNA was extracted and analyzed after 5 h of growth of SM101 cultures in the indicated medium with or without added Pi as previously described (11). The data from a representative experiment are shown.

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  • TABLE 1.

    Sporulation of C. perfringens in DSMM, with or without added phosphate

    Added Pi (mM)Initial pHFinal pHNo. of viable cells/mlNo. of spores/ml% Sporulationa
    6.625.044.7 × 108<10<0.000002
    16.655.052.5 × 108<10<0.000004
    26.665.053.0 × 108<10<0.000003
    36.685.071.0 × 1083.2 × 1010.00003
    56.735.123.7 × 1072.7 × 1067.29
    76.895.141.9 × 1074.6 × 10624.21
    106.915.211.3 × 1076.3 × 10648.46
    157.035.534.5 × 1073.0 × 10766.66
    207.175.803.9 × 1073.3 × 10784.61
    257.296.121.3 × 1071.2 × 10792.30
    307.356.302.0 × 1072.0 × 107100
    357.396.401.4 × 1071.4 × 107100
    407.506.532.1 × 1072.1 × 107100
    457.556.613.6 × 1073.3 × 10791.66
    507.596.694.2 × 1073.1 × 10773.80
    607.616.838.0 × 1061.2 × 10615.00
    707.687.002.5 × 1066.8 × 1042.72
    807.807.179.3 × 1051.3 × 1030.14
    907.937.214.5 × 1052.8 × 1020.06
    1007.987.303.1 × 1051.7 × 1010.005
    • ↵ a The percentage of sporulation was calculated as follows: (no. of heat-resistant spores/no. of viable cells) × 100.

  • TABLE 2.

    Phosphate-induced sporulation in nonsporulation media

    Growth mediumaNo. of viable cells/mlNo. of spores/mlInduction (n-fold)
    DSMM1.3 × 1080>1,900,000
    DSMM+Pi2.0 × 1071.9 × 107
    FT3.4 × 1070>1,000
    FT+Pi2.0 × 1061.0 × 104
    TY3.0 × 1073015,333
    TY+Pi2.0 × 1064.6 × 105
    • ↵ a Pi was used at a concentration of 35 mM.

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Inorganic Phosphate Induces Spore Morphogenesis and Enterotoxin Production in the Intestinal Pathogen Clostridium perfringens
Valeria A. Philippe, Marcelo B. Méndez, I-Hsiu Huang, Lelia M. Orsaria, Mahfuzur R. Sarker, Roberto R. Grau
Infection and Immunity May 2006, 74 (6) 3651-3656; DOI: 10.1128/IAI.02090-05

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Inorganic Phosphate Induces Spore Morphogenesis and Enterotoxin Production in the Intestinal Pathogen Clostridium perfringens
Valeria A. Philippe, Marcelo B. Méndez, I-Hsiu Huang, Lelia M. Orsaria, Mahfuzur R. Sarker, Roberto R. Grau
Infection and Immunity May 2006, 74 (6) 3651-3656; DOI: 10.1128/IAI.02090-05
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KEYWORDS

Clostridium perfringens
enterotoxins
Phosphates

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