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MOLECULAR AND CELLULAR PATHOGENESIS

Cytotoxic Cell Vacuolating Activity from Vibrio cholerae Hemolysin

Ana Coelho, João R. C. Andrade, Ana Carolina P. Vicente, Victor J. Dirita
Ana Coelho
Departamento de Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ21944-970,
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João R. C. Andrade
Serviço de Microbiologia e Immunologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ20551-030, and
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Ana Carolina P. Vicente
Departamento de Genética, Instituto Oswaldo Cruz, Rio de Janeiro, RJ21045-100, Brazil, and
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Victor J. Dirita
Unit for Laboratory Animal Medicine and Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109
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DOI: 10.1128/IAI.68.3.1700-1705.2000
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    Fig. 1.

    Vacuolation of mammalian cells caused by a cytotoxin secreted by V. cholerae. (A) Control normal Vero cells. (B) Vacuolated cells after treatment with the supernatant of a V. cholerae strain (Amazonia), seen by phase-contrast microscopy (magnification, ×320). (C andD) Vacuolated cells after a neutral red uptake assay, done after 24 h of incubation with the cytotoxin. The weak base neutral red is taken up by the vacuoles.

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  • Table 1.

    Characteristics and effect on Vero cells of strains used

    StrainDescriptionaVcVacTiterbNo vacuolation
    V. cholerae
     O1 Amazonia20/20c
      FG1066, FG1068 and 18 othersAmazonas, Brazil, 91-92+
      FG1066S, 1/640; C, 1/10,240
     O1 El Tor5/1813/18
      E7946Ogawa, Bahrain, 78S, C
      ANCO223Ogawa, PeruS, C
      0872Inaba, Peru+S, 1/120; C, 1/2,560
      N16961Inaba, Bangladesh, 75+S, 1/30; C, 1/320
      N16117Ogawa, BangladeshS, C
      P27459Inaba, Bangladesh, 76S, C
      4 strainsBrazil, 92-94S, C
      T19479Bangladesh, 79S, C
      26-3Ogawa, Philippines, 61+S, 1/60; C, 1/480
      30167Bangladesh, 76S, C
      62746Bangladesh, 76S, C
      N.2Inaba, China, 86+S, 1/20; C, 1/160
      N.5Ogawa, China, 77+S, 1/960; C, >1/2,560
      62-6-91TanzaniaS, C
      LA-M-644NigeriaS, C
     O1 Gulf of Mexico El Tor3/3
      E506Texas+S, 1/20; C, 1/80
      SGN7277Sewage, Louisiana, 80+S, 1/20
      4808-78Patient, Louisiana, 78+S, 1/20
     O1 classical6/6
      0395India, 64S, C
      AMS 20-A-73 (VRL3)China, 45S, C
      N32089Bangladesh, 82S, C
      N19073Bangladesh, 82S, C
      N19812Bangladesh, 82S, C
      N22646Bangladesh, 82S, C
     Environmental, non-O1, non-01397/10 3/10
      FG1130Water, RJ, Brazil, 97+S, 1/320
      FG1131Water, RJ, Brazil, 97+S, 1/1920
      FG1132Mollusk, RJ, Brazil, 97S, C
      FG1133Mollusk, RJ, Brazil, 97S, C
      FG1134Mollusk, RJ, Brazil, 97+S, 1/20
      FG1138Mollusk, RJ, Brazil, 95+S, 1/80
      FG1139Crab, RJ, Brazil, 96S, C
      FG1140Crab, RJ, Brazil, 97+S, 1/30
      FG1135Water, RJ, Brazil, 97+S, 1/40
      FG1137Crab, RJ, Brazil, 95+S, 1/240
    E. colid
     DH5α
     DH5αλpirFrom A. Camilli, Tufts University
     EK322MM294/pRK2013
     VJ737MC4100/pKAS32 from R. Taylor, Dartmouth University
     FG1155DH5αλpir/pFG300, this work
     FG1221DH5α/pFG350, this work
    Other O1 V. cholerae strains
     JBK56Inaba, derived from N16961, Δctx Apr
     JBK70Inaba, derived from N16961, Δctx Hgr
     CVD104Inaba, N16961 Δctx ΔhlyA
     CVD113Ogawa, E7946 Δvirulence cassette ΔhlyA::mer ctxB, Δlec
     FG1106FG1066 Smr, this work
     FG1153FG1106 hlyA::pFG300, Apr, this work
     FG1173Hemolytic revertant of FG1153, this work
     FG1167Smr revertant of FG1153, this work
     FG1229FG1153/pFG350, this work
    • ↵a Numerals indicate years (91-92 denotes 1991 to 1992, etc.) of isolation at the location given.

    • ↵b S, filtered supernatant of culture; C, 80-fold volume concentrate prepared from S by precipitating with 50% ammonium sulfate.

    • ↵c Fraction of VcVac-positive strains in each group.

    • ↵d Either strains have not been tested on cells or their results are presented in a separate table.

  • Table 2.

    Comparison of vacuolating activities of Amazonia and vaccine strains derived from the El Tor strain N16961

    FractionDilution yielding maximum vacuolating activity
    FG1066 (Amazonia)N16961 (El Tor, wild type)JBK56 (CT− Apr)JBK70 (CT−Hgr)CVD104 (CT− HlyA−)
    Supernatant1/6401/201/201/20<1/20
    Concentrate1/51201/3201/3201/320<1/20
  • Table 3.

    Comparison of vacuolating cytotoxic activities from various strains in hlyA inactivation, reversion, cloning, and complementation experimentsa

    FractionDilution yielding maximum vacuolating activity
    FG1106FG1153FG1173FG1167DH5αFG1221FG1229
    Supernatant1/212<1/201/1621/125<1/20<1/201/853
    Concentrate1/4,096<1/201/7,6801/5,120<1/101/301/40,960
    • ↵a See Table 1 for descriptions of the strains used.

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Cytotoxic Cell Vacuolating Activity from Vibrio cholerae Hemolysin
Ana Coelho, João R. C. Andrade, Ana Carolina P. Vicente, Victor J. Dirita
Infection and Immunity Mar 2000, 68 (3) 1700-1705; DOI: 10.1128/IAI.68.3.1700-1705.2000

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Cytotoxic Cell Vacuolating Activity from Vibrio cholerae Hemolysin
Ana Coelho, João R. C. Andrade, Ana Carolina P. Vicente, Victor J. Dirita
Infection and Immunity Mar 2000, 68 (3) 1700-1705; DOI: 10.1128/IAI.68.3.1700-1705.2000
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KEYWORDS

cytotoxins
Escherichia coli Proteins
Hemolysin Proteins
macrolides
Vacuoles
Vibrio cholerae

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