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Infection and Immunity, April 2000, p. 1928-1933, Vol. 68, No. 4
National Institute of Cholera and Enteric
Diseases, Calcutta, India1; Department
of Molecular Microbiology, Washington University School of Medicine,
St. Louis, Missouri2; Escuela Nacional
de Ciencias Biologicas del Instituto Politecnico Nacional, Carpio y
Plan de Ayala, Mexico City, Mexico 113403;
and National Institute of Infectious Diseases, Shinjuku-ku,
Tokyo, Japan4
Received 27 September 1999/Returned for modification 4 November
1999/Accepted 22 December 1999
Culture supernatants of nontoxigenic nonepidemic clinical strains
of Vibrio cholerae belonging to diverse serogroups were found to induce vacuolation of nonconfluent HeLa cells. The vacuoles became prominent 18 h after introduction of culture supernatant, and vacuolated cells survived for 48 h and then died. Only a
fraction of the vacuolated cells took up neutral red dye, implying that there were differences in the vacuolar microenvironment. Further tests
showed that the factor responsible for vacuolation was heat labile and
proteinaceous. Vacuolating activity was completely neutralized by
antibody to hemolysin of V. cholerae but not by antibody to
vacuolating cytotoxin of Helicobacter pylori. Partial purification of the vacuolating factor led to elution of fractions, which showed both hemolytic and vacuolating activity. PCR amplification and cloning of the hemolysin structural gene (hlyA) into
Escherichia coli DH5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cell Vacuolation, a Manifestation of the El Tor
Hemolysin of Vibrio cholerae
led to isolation of clones
producing cell vacuolating factor in a cell-associated form. Further, a
null insertion mutation in the hlyA gene of a
high-vacuolating-factor-producing strain led to complete abolition of
both cell vacuolating and hemolytic activities. These analyses
establish vacuolation as a potentially important but previously
unrecognized property of V. cholerae El Tor hemolysin.
*
Corresponding author. Mailing address: National
Institute of Cholera and Enteric Diseases, P-33 C.I.T. Rd., Scheme XM,
Calcutta 700010, India. Phone: 350-4598. Fax: 91-33-350-5066/353-2524. E-mail: gbnair{at}vsnl.com.
Infection and Immunity, April 2000, p. 1928-1933, Vol. 68, No. 4
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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