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Infection and Immunity, January 1999, p. 466-467, Vol. 67, No. 1
0019-9567/99/$00.00+0
LETTERS TO THE EDITOR
The Cytotoxic Enterotoxin of Aeromonas hydrophila Is
Aerolysin
 |
LETTER |
The channel-forming toxin aerolysin was identified 25 years ago by
Bernheimer and Avigad (1), who gave the protein its name.
Aerolysin was purified by Buckley et al. (2), and its structural gene, named aerA, was cloned and sequenced almost
simultaneously by two groups (6, 7). Since then more than 50 articles describing the expression, secretion, and properties of
aerolysin have appeared, and aerolysin has become one of the
best-characterized bacterial channel-forming toxins. Virtually all of
the available evidence indicates that aerolysin kills cells by forming
discrete channels in their plasma membranes (for a recent review, see
reference 8). Although it is certainly cytotoxic, it
has not been shown to directly alter cyclic nucleotide levels and
therefore does not satisfy the definition of an enterotoxin.
Two articles in Infection and Immunity from the group of
Chopra have described the purification and mechanism of action
(5), and the role in Aeromonas-mediated infection
(9), of a cytotoxic enterotoxin from A. hydrophila. The authors state that their protein (referred to as
"Act") is "an aerolysin-related toxin." In fact, this protein
is aerolysin. Its amino acid sequence is essentially the same as the
six other aerolysin sequences (from different Aeromonas
strains or species) retrieved by a BLAST search (five of these are
referred to as aerolysin and the other as a hemolysin). For example,
416 (97%) of the 427 amino acids in the active, biologically relevant
form of the protein described by Chopra's group (accession no.
2126218) are the same as those in the first aerolysin sequence which
was obtained (accession no. 113485), and 6 of the 11 differences are conservative.
All of the properties of the protein described by the Chopra group have
previously been described for aerolysin, with one minor apparent
exception. In one article (4) it is pointed out that the
results of mutagenesis of the cytotoxic enterotoxin do not completely
correspond to results reported for aerolysin. However, the authors were
not working with purified proteins in this study, nor was there
evidence that they had confirmed each of the changes they attempted.
The fact that Chopra et al. do not refer to their protein as aerolysin
is not productive. Medline searches using aerolysin as a key word do
not locate all of their relevant articles, because aerolysin does not
appear in the titles, abstracts, or key words. It is difficult to
compare their findings with those of others and to determine if data
are being duplicated. This is most recently illustrated in their last
Infection and Immunity article, in which they describe the
testing in a lethal mouse assay of mutants which do not produce
cytotoxic enterotoxin (9), comparable to work done 10 years
ago with the same results (3). We believe that the authors
should use the word aerolysin to describe their protein, as all other
groups have done, or they should provide convincing evidence that they
are working with a different toxin.
| |
REFERENCES |
| 1.
|
Bernheimer, A. W., and L. S. Avigad.
1974.
Partial characterization of aerolysin, a lytic exotoxin from Aeromonas hydrophila.
Infect. Immun.
9:1016-1021[Abstract/Free Full Text].
|
| 2.
|
Buckley, J. T.,
L. N. Halasa,
K. D. Lund, and S. MacIntyre.
1981.
Purification and some properties of the hemolytic toxin aerolysin.
Can. J. Biochem.
59:430-436[Medline].
|
| 3.
|
Chakraborty, T.,
B. Huhle,
H. Hof,
H. Bergbauer, and W. Goebel.
1987.
Marker exchange mutagenesis of the aerolysin determinant in Aeromonas hydrophila demonstrates the role of aerolysin in A. hydrophila-associated systemic infections.
Infect. Immun.
55:2274-2280[Abstract/Free Full Text].
|
| 4.
|
Ferguson, M. R.,
X.-J. Xu,
C. W. Houston,
J. W. Peterson, and A. K. Chopra.
1995.
Amino-acid residues involved in biological functions of the cytolytic enterotoxin from Aeromonas hydrophila.
Gene
156:79-83[Medline].
|
| 5.
|
Ferguson, M. R.,
X.-J. Xu,
C. W. Houston,
J. W. Peterson,
D. H. Coppenhaver,
V. L. Popov, and A. K. Chopra.
1997.
Hyperproduction, purification, and mechanism of action of the cytotoxic enterotoxin produced by Aeromonas hydrophila.
Infect. Immun.
65:4299-4308[Abstract].
|
| 6.
|
Howard, S. P., and J. T. Buckley.
1987.
Nucleotide sequence of the gene for the hemolytic toxin aerolysin.
J. Bacteriol.
169:2869-2871[Abstract/Free Full Text].
|
| 7.
|
Husslein, V.,
B. Huhle,
T. Jarchau,
R. Lurz,
W. Goebel, and T. Chakraborty.
1988.
Nucleotide sequence and transcriptional analysis of the aerCaerA region of Aeromonas sobria encoding aerolysin and its regulatory region.
Mol. Microbiol.
2:507-517[Medline].
|
| 8.
|
Parker, M. W.,
F. G. van der Goot, and J. T. Buckley.
1996.
Aerolysin the ins and outs of a channel-forming protein.
Mol. Microbiol.
19:205-212[Medline].
|
| 9.
|
Xu, X.-J.,
M. R. Ferguson,
V. L. Popov,
C. W. Houston,
J. W. Peterson, and A. K. Chopra.
1998.
Role of a cytotoxic enterotoxin in Aeromonas-mediated infections: development of transposon and isogenic mutants.
Infect. Immun.
66:3501-3509[Abstract/Free Full Text].
|
| | | | |
J.
Thomas Buckley
Department of Biochemistry and
Microbiology University of Victoria Box
3055 Victoria, British Columbia Canada V8W 3P6
|
| | | | |
S. Peter Howard
Department of Biology
University of Regina Regina, Saskatchewan
Canada S4S 0A2
|
| |
AUTHORS' REPLY |
Enterotoxins have been classified as cytotonic and cytotoxic
(10) and cause fluid secretory responses in animals
irrespective of their capacity to evoke cyclic nucleotide levels in
cells. If the statement of Buckley and Howard is true, then heat-stable enterotoxin (STb) of Escherichia coli should not be referred
to as an enterotoxin as it does not alter cyclic nucleotide levels in
cells (14), and the same is true for many other enterotoxins (12). In 1992, we presented the amino acid (aa) sequence of a cytotoxic enterotoxin (Act) from Aeromonas hydrophila at
the American Society for Microbiology meeting, and the sequence was sent to Dr. Buckley at his request. Within aa residues 449 to 462, there was only a 21 to 36% homology between Act and other two
aerolysins (2), and within aa residues 482 to 493, there were marked sequence differences (8% homology) between our Act and
aerolysin isolated by Buckley's group (2, 8). Since then,
they have corrected the sequence in this region (aa 482 to 493)
compared to their originally published sequence (8), and
consequently, it matched our sequence (2, 13).
In our recent papers (4, 15), we provided new information
about Act that was not known earlier for aerolysin. This information includes, but is not limited to, cholesterol as one of the receptors for the toxin; the inability of Act, unlike aerolysin (6,
7), to bind to glycophorin; isolation and characterization of
naturally occurring toxin-deficient mutants of Aeromonas;
and generation of potential regulatory mutants of Aeromonas
with altered toxin activity. Consistent with these findings, Buckley
and Howard also indicate in their letter that the results of
site-directed mutagenesis of Act do not completely correspond to
results reported for aerolysin. We believe that these results on
mutagenesis are significant and not "one minor apparent exception"
because some of the selected mutated toxin proteins purified thus far
exhibited the same biological activities as crude toxin preparations.
All of the aa changes were confirmed by sequence analysis
(5). We have referred to our toxin as a cytotoxic
enterotoxin for a number of years based on the hemolytic, cytotoxic,
and enterotoxic nature of this toxin. The aerolysin gene sequenced by
Chakraborty's group from Aeromonas sobria (now A. trota) (9) exhibited 76% homology with that of our
act (2) and the aerolysin gene sequenced by
Buckley's group (8). Although Chakraborty et al.
(1) generated an aerolysin gene-deficient mutant of A. hydrophila (now A. trota), our studies indicated that
Act and the aerolysin isolated by Buckley's group were not identical
to A. trota aerolysin based on differential neutralization
by a specific monoclonal antibody (3). Therefore, it was
important to delete the act gene from an authentic strain of
A. hydrophila, particularly as Kuhn et al. (11)
reported that A. hydrophila type HG1/BD-2 may be able to
produce diarrhea in humans. In our papers, we have indicated that Act
is an aerolysin-like molecule because it differs in some respects from
aerolysin. Other investigators have named their toxins "aerolysin"
primarily based on DNA sequence analysis. However, our studies are
based on molecular, biochemical, and biological properties of the
toxin. Currently, we strongly feel that we should refer to our toxin as
a cytotoxic enterotoxin; however, we will be careful in our future
publications so that other investigators will be able to retrieve our
relevant articles in Medline searches using aerolysin as a key word.
| |
REFERENCES |
| 1.
|
Chakraborty, T.,
B. Huhle,
H. Hof,
H. Bergbauer, and W. Goebel.
1987.
Marker exchange mutagenesis of the aerolysin determinant in Aeromonas hydrophila demonstrates the role of aerolysin in A. hydrophila-associated systemic infections.
Infect. Immun.
55:2274-2280.
|
| 2.
|
Chopra, A. K.,
C. W. Houston,
J. W. Peterson, and G.-F. Jin.
1993.
Cloning, expression, and sequence analysis of a cytolytic enterotoxin gene from Aeromonas hydrophila.
Can. J. Microbiol.
39:513-523[Medline].
|
| 3.
|
Chopra, A. K.,
C. W. Houston, and A. Kurosky.
1991.
Genetic variation in related cytolytic toxins produced by different species of Aeromonas.
FEMS Microbiol. Lett.
78:231-238.
|
| 4.
|
Ferguson, M. R.,
X.-J. Xu,
C. W. Houston,
J. W. Peterson,
D. H. Coppenhaver,
V. L. Popov, and A. K. Chopra.
1997.
Hyperproduction, purification, and mechanism of action of the cytototoxic enterotoxin produced by Aeromonas hydrophila.
Infect. Immun.
65:4299-4308.
|
| 5.
|
Ferguson, M. R.,
X.-J. Xu,
C. W. Houston,
J. W. Peterson, and A. K. Chopra.
1995.
Amino-acid residues involved in biological functions of the cytolytic enterotoxin from Aeromonas hydrophila.
Gene
156:79-83.
|
| 6.
|
Garland, W. J., and J. T. Buckley.
1988.
The cytolytic toxin aerolysin must aggregate to disrupt erythrocyte membranes, and aggregation is stimulated by human glycophorin.
Infect. Immun.
56:1249-1253[Abstract/Free Full Text].
|
| 7.
|
Herman, J. G.,
H. U. Wilmsen,
S. Cowell,
H. Schindler, and J. T. Buckley.
1994.
Partial purification of the rat erythrocyte receptor for channel-forming toxin aerolysin and reconstitution into planner lipid bilayers.
Mol. Microbiol.
14:1093-1101[Medline].
|
| 8.
|
Howard, S. P.,
W. J. Garland,
M. J. Green, and J. T. Buckley.
1987.
Nucleotide sequence of the gene for the hole-forming toxin aerolysin of Aeromonas hydrophila.
J. Bacteriol.
169:2869-2871.
|
| 9.
|
Husslein, V.,
B. Huhle,
T. Jarchau,
R. Lurz,
W. Goebel, and T. Chakraborty.
1988.
Nucleotide sequence and transcriptional analysis of the aerCaerA region of Aeromonas sobria encoding aerolysin and its regulatory region.
Mol. Microbiol.
2:507-517.
|
| 10.
|
Keusch, G. T., and S. T. Donta.
1975.
Classification of enterotoxins on the basis of activity in cell culture.
J. Infect. Dis.
131:58-63[Medline].
|
| 11.
|
Kühn, I.,
M. J. Albert,
M. Ansaruzzaman,
N. A. Bhuiyan,
S. A. Alabi,
M. S. Islam,
P. K. B. Neogi,
G. Huys,
P. Janssen,
K. Kersters, and R. Möllby.
1997.
Characterization of Aeromonas spp. isolated from humans with diarrhea, from healthy controls, and from surface water in Bangladesh.
J. Clin. Microbiol.
35:369-373[Abstract].
|
| 12.
|
Salyers, A. A., and D. D. Whitt.
1994.
Bacterial pathogenesis: a molecular approach, p. 131-140.
ASM Press, Washington, D.C.
|
| 13.
|
van der Goot, F. G.,
J. Lakey,
F. Pattus,
C. M. Kay,
O. Sorokine,
A. Van Dorsselaer, and J. T. Buckley.
1992.
Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation.
Biochemistry
31:8566-8570[Medline].
|
| 14.
|
Weikel, C. S., and R. C. Guerrant.
1985.
ST enterotoxin of Escherichia coli: cyclic nucleotide-independent secretion, p. 94-115.
In
R. Evered, and J. Whelan (ed.), Microbial toxins and diarrheal disease. Pitman, London, United Kingdom.
|
| 15.
|
Xu, X.-J.,
M. R. Ferguson,
V. L. Popov,
C. W. Houston,
J. W. Peterson, and A. K. Chopra.
1998.
Role of a cytotoxic enterotoxin in Aeromonas-mediated infections: development of transposon and isogenic mutants.
Infect. Immun.
66:3501-3509.
|
| | | | |
A. K. Chopra
C. W. Houston
Department of Microbiology and Immunology
University of Texas Medical Branch
Galveston, Texas 77555-1070
|
Infection and Immunity, January 1999, p. 466-467, Vol. 67, No. 1
0019-9567/99/$00.00+0
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