INFECTION AND IMMUNITY,
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
July 1998, p. 3149–3154Vol. 66, No. 7
In Vitro Effects of a High-Molecular-Weight Heat-Labile
Enterotoxin from Enteroaggregative Escherichia coli
FERNANDO NAVARRO-GARCI´A,1,2,3* CARLOS ESLAVA,1JORGE M. VILLASECA,1
RUBE´N LO´PEZ-REVILLA,2JOHN R. CZECZULIN,3S. SRINIVAS,4
JAMES P. NATARO,3AND ALEJANDRO CRAVIOTO1
Department of Public Health, Faculty of Medicine, UNAM, 04510 Mexico DF,1and Department
of Cell Biology, CINVESTAV-IPN, 07000 Mexico DF,2Mexico, and Center for Vaccine
Development, Department of Pediatrics,3and Veterinary Resources,4University
of Maryland School of Medicine, Baltimore, Maryland 21201
Received 9 February 1998/Returned for modification 25 March 1998/Accepted 20 April 1998
The pathogenic mechanisms of enteroaggregative Escherichia coli (EAggEC) infection are not fully eluci-
dated. In this work we show that an ammonium sulfate precipitate of culture supernatant of EAggEC strain
049766 increased the potential difference (PD) and the short-circuit current (Isc) in rat jejunal preparations
mounted in Ussing chambers. The precipitate contained two major proteins of 108 and 116 kDa, which were
partially copurified by chromatography in DEAE-cellulose. This chromatographic fraction (peak I) increased
jejunal PD and Isc in a dose-dependent manner, accompanied by a decrease in tissue electrical resistance.
These effects were inhibited by incubation of peak I at 75°C for 15 min or for 1 h with proteinase K at 37°C.
Rabbit polyclonal antibodies against peak I containing both the 108- and 116-kDa proteins inhibited the en-
terotoxic effect. Specific polyclonal antibodies raised against the 108-kDa but not against the 116-kDa protein
inhibited the enterotoxic effect, suggesting that the 108-kDa protein is the active toxic species. Moreover,
another EAggEC strain (065126) producing the 116-kDa protein but not the 108-kDa protein had no effect on
rat jejunal mucosa in the Ussing chamber. The >100-kDa fraction derived from prototype EAggEC strain 042,
which also expressed both 108- and 116-kDa proteins, also produced an enterotoxic effect on rat jejunal
preparations in Ussing chambers; however, the same strain cured of its 65-MDa adherence plasmid did not.
A subclone derived from the 65-MDa plasmid expressing the 108-kDa toxin (and not the 116-kDa protein)
elicited rises in Isc. Tissue exposed to any preparation containing the 108-kDa toxin exhibited similar histo-
pathologic changes, characterized by increased mucus release, exfoliation of cells, and development of crypt
abscesses. Our data suggest that some EAggEC strains produce a ca. 108-kDa enterotoxin/cytotoxin which is
encoded on the large virulence plasmid.
Enteroaggregative Escherichia coli (EAggEC) has been as-
sociated with persistent diarrhea in young children (3, 5, 15,
25), especially in developing countries. Most EAggEC strains
harbor a 65-MDa plasmid (called pAA), which is required for
expression of aggregative adherence fimbriae (AAFs). These
structures mediate the defining aggregative adherence (AA)
phenotype to HEp-2 cells (14, 15, 21) as well as adherence to
the colonic mucosa (7). The pAA plasmid is also required for
the development of mucosal damage in in vitro models (10,
Clinical, volunteer, and animal model studies suggest that
EAggEC diarrhea may be due to a secretogenic enterotoxin
(3, 13, 25). When tested in Ussing chambers, filtrates from
EAggEC strain 17-2 produced an increase in potential differ-
ence (PD) and short-circuit current (Isc), attributed by Sa-
varino et al. (20) to a heat-stable, plasmid-encoded enterotoxin
of less than 10 kDa in molecular mass (EAST1). No data yet
exist to support a role for EAST1 in EAggEC diarrhea.
Nataro et al. (13) reported that EAggEC strain 042 (sero-
type O44:H18) caused significant diarrhea in three of five adult
volunteers, whereas strains 17-2, 34b, and JM221 (EAggEC
of serotypes O3:H2, O?:H?, and O92:H33, respectively) did
not induce enteric symptoms. Except for 042, each of these
strains expressed AAF/I fimbriae, while EAST1 was produced
by strains 042 and 17-2 but not by strain 34b or JM221 (13).
Mathewson et al. (12) had previously reported that strain
JM221 caused mild diarrhea in some adult volunteers, and
Tzipori et al. (23) had shown that strains 17-2 and JM221 were
able to cause diarrhea in gnotobiotic piglets. The basis for this
strain heterogeneity has not been determined, and these data
suggest the presence of unrecognized virulence factors.
A role for cytotoxins in EAggEC disease has been suggested
by in vivo and in vitro models (10, 16, 24), which exhibit dam-
age to intestinal epithelium. A candidate cytotoxin has been
identified by Eslava et al. (8), as sera from children in a Mex-
ican EAggEC outbreak consistently recognized two proteins of
108 and 116 kDa obtained from ammonium sulfate precipi-
tates of EAggEC culture supernatants. Together these pro-
teins elicited hemorrhagic and necrotic lesions in rat ileal loops
(8). In this communication, we report that a fraction containing
the 108- and 116-kDa proteins purified from EAggEC 049766,
the strain implicated in the Mexican outbreak, is able to cause
enterotoxic and cytotoxic effects on rat jejunal tissue mounted
in Ussing chambers. We have identified the toxin moiety as the
108-kDa protein and have localized it to the pAA plasmid.
MATERIALS AND METHODS
Bacterial strains. EAggEC strain 049766, implicated in an outbreak of per-
sistent diarrhea in Mexican infants, has been characterized as belonging to
serotype O?:H10 and is capable of attaching with an aggregative pattern to HEp-
2 cells (6, 15). EAggEC strain 065126 was isolated from a Mexican child with
diarrhea. Strain 042 was isolated from a child with diarrhea in Lima, Peru, in
* Corresponding author. Mailing address: Department of Public
Health, Faculty of Medicine, UNAM, Ap. Postal 70-443, 04510 Mexico
DF, Mexico. Phone: (525) 622-0822. Fax: (525) 622-0827. E-mail:
1983; this strain has been shown to cause diarrhea in adult volunteers (13).
Strains 049766, 065126, and 042 hybridize with the EAggEC-specific (AA) probe
(2). Plasmid-cured 042 has been described previously (16). E. coli strain K-12 was
used as a control in Ussing chamber experiments; E. coli HB101 was used as a
host for cloning experiments.
Preparation of protein fractions. Culture conditions and preparation of pre-
cipitates were performed as follows. EAggEC strain 049766 was grown overnight
at 37°C in 200 ml of Luria broth (LB). After centrifugation at 12,000 ? g for 10
min, supernatants were precipitated with 60% saturated ammonium sulfate for
18 h at 4°C, collected by centrifugation, dissolved with 0.07 M potassium phos-
phate buffer (pH 8.2), and dialyzed for 4 days against the same buffer. Protein
concentration was determined by the method of Bradford (4). Purification of
EAggEC-secreted proteins was obtained by further precipitation of protein sus-
pensions in 0.07 M potassium phosphate buffer with 1.75 M K2HPO4, dialyzed at
4°C against Tris-EDTA buffer (0.05 M Tris-0.1 M EDTA [pH 8.0]) and eluted
with the same buffer from a DEAE-cellulose column.
For the neutralization experiments in the Ussing chamber, performed at the
Center for Vaccine Development at Baltimore, EAggEC strain 049766 was
grown overnight at 37°C in 100 ml of LB. After centrifugation at 12,000 ? g for
10 min, supernatants were concentrated and size fractionated by passage through
Biomax-50 Ultrafree filters (Millipore, Bedford, Mass.) according to the manu-
Protein electrophoresis and immunologic methods. Proteins present in pre-
cipitated culture supernatants from the DEAE chromatography fractions were
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) by the method of Laemmli (11) under reducing conditions (boiling for 5
min in the presence of mercaptoethanol). Proteins separated by SDS-PAGE
were transferred to nitrocellulose BA85 membranes (Schleicher & Schuell,
Keene, N.H.) by the method of Towbin et al. (22). The membranes were incu-
bated with rabbit antibodies generated in our laboratory against the 108- and
116-kDa proteins (diluted 1:100) or with sera from children in the Mexican
EAggEC outbreak (diluted 1:25). Immunostaining was performed with peroxi-
dase-labeled polyclonal antibodies against rabbit immunoglobulins (dilution
1:5,000) and developed with 4-chloronaphthol by standard methods (22). The
antibodies against each of the 108- and 116-kDa proteins were elicited by excis-
ing proteins from polyacrylamide gels and injecting the gel slices subcutaneously
into rabbits in two doses, 2 weeks apart. The antibody responses and specificity
were determined by immunoblotting. The gamma fractions from the antisera
were diluted 1:25 prior to use in Ussing chamber experiments.
Electrophysiologic measurements in rat jejunum. Ussing chamber experi-
ments in Mexico City were performed as described previously (18, 19). Jejunal
segments removed from adult male Sprague-Dawley rats under sodium pento-
barbital anesthesia were placed in ice-cold Ringer’s solution for mammals and
gassed with an O2-CO2(95%:5%) mixture. The excised segments were cut open
along the mesenteric border, washed with cold Ringer’s solution, divided into
two fragments (experimental and control), and mounted between the circular
openings (6-mm diameter, 0.28 cm2) of two adjacent Ussing hemichambers.
Each hemichamber was filled with 10 ml of Ringer’s solution and kept at 37°C
under constant O2-CO2bubbling. Transmural PD and Isc were recorded at
1-min intervals by means of a voltage clamp apparatus. Samples containing 1.5 to
25 ?g of precipitate per ml were diluted with Ringer’s solution at 37°C and added
to the mucosal hemichamber of rat jejunum preparations after 10 min of equil-
ibration, and both hemichambers were gassed with O2-CO2. Transmural resis-
tance (R) values were obtained from PD and Isc values by using Ohm’s law.
Statistical analyses were performed with Student’s t test on data recorded from
at least four experiments.
Ussing chamber experiments in which enterotoxic activity was inhibited by
antibodies against either the 108- or 116-kDa protein were performed at the
University of Maryland by methods previously described (9). Six pieces of rat
jejunum were mounted in Ussing chambers; a known positive control and ap-
propriate negative control were always assayed in parallel with the test samples
(culture filtrates of strain 049766 with or without antibodies), using the same rat
tissue. PD was measured at intervals, and total tissue conductance and Isc were
Supernatants from strains 049766, 042, 065126, and HB101(pJPN201) used in
Ussing chamber experiments were concentrated 100? and size fractionated by
using Biomax Ultrafree filters (100-kDa cutoff; Millipore) according to the man-
ufacturer’s instructions. Neutralization of the electrophysiological effects of
EAggEC proteins was tested by using aliquots containing 25 ?g of partially
purified EAggEC proteins that were either heat treated at 75°C for 15 min or
incubated with proteinase K (200 ?g/ml) at 37°C for 1 h before being added to
the luminal side of jejunal preparations mounted in Ussing chambers. To test the
FIG. 1. SDS-PAGE characterization of protein fractions (40 ?g per lane)
from EAggEC strain 049766. E. coli K-12 was used as a control. The crude 60%
(NH4)2SO4-precipitated supernatant (lane C) of 049766 produced several pro-
teins, including those at 108 and 116 kDa. The high-molecular-weight fraction
was partially purified by reprecipitation with 1.75 M K2HPO4(lane B) and
chromatography through DEAE-cellulose (peak I, lane D). Concentrated super-
natant of E. coli K-12 (lane A) did not reveal secreted proteins in the range of
108 to 116 kDa.
FIG. 2. Time course of PD, Isc, and R values of rat jejunum preparations
exposed to 60% (NH4)2SO4-precipitated supernatants from cultures of EAggEC
strain 049766 or E. coli K-12. Twenty-five micrograms of protein was used from
concentrated supernatants or 25 ?l of uninoculated LB. The symbols represent
the mean values of experiments performed on four different animals.
TABLE 1. Increase in PD and Isc after addition of various doses of
partially purified 108- and 116-kDa EAggEC-secreted proteins
in rat jejunum strips mounted in Ussing chambersa
Time of maximum
aAverage values for each dose (n ? 4).
bPD and Isc started to increase 20 min after addition of protein preparations.
3150 NAVARRO-GARCI´A ET AL.INFECT. IMMUN.
inhibitory effects of different antibodies, 25 ?g of partially purified EAggEC
proteins was preincubated for 20 min at room temperature with rabbit polyclonal
antibodies directed against the 108- or 116-kDa protein (diluted 1:25) before
being added to the luminal hemichamber.
To assess the integrity of intestinal preparations at the end of the electrophysi-
ologic experiments, samples were removed from Ussing chambers and fixed for
1 h in 10% formalin, embedded in paraffin, and cut into 4- to 6-?m sections that
were stained with hematoxylin and eosin (18) and examined under light micros-
Ussing chamber effects of 108- and 116-kDa supernatant
proteins. Precipitation of EAggEC 049766 culture superna-
tants using 60% saturated (NH4)SO4yielded several proteins,
most prominent of which were 108- and 116-kDa species (Fig.
1, lane C) that were absent from precipitates of culture su-
pernatants of an E. coli K-12 strain (lane A). Addition of the
precipitates obtained from the 60% saturated (NH4)2SO4su-
pernatant of 049766 (lane C) to the mucosal hemichamber of
rat jejunum strips mounted in Ussing chambers evoked a sig-
nificant increase of PD and Isc while producing a decrease of
R (Fig. 2). Rises in PD and Isc began approximately 20 min
after addition of culture supernatants. PD and Isc rose from
0.5 to 1.04 mV and from 5.1 to 16.7 ?A/cm2, respectively, while
the R values decreased about 50% (P ? 2.8 ? 10?8), from 102
to 58 ?cm2. Maximum increases were attained approximately
95 min after inoculation. Precipitates from culture superna-
tants of an E. coli K-12 and from uninoculated broth had no
effect on jejunal preparations from the same animals (Fig. 2).
These data suggest that the supernatant of strain 049766 con-
tains an enterotoxin.
To identify the protein conferring the enterotoxic effect,
FIG. 3. SDS-PAGE (A) and Western immunoblotting (B to D) of ?100-kDa fractions from supernatants of strains 049766 (lanes a), 065126 (lanes b), 042 (lanes
c), and HB101(pJPN201) (lanes d). In panel B, Western blots in lanes a to d are reacted with anti-peak I antibodies, and those in lanes e and f are reacted with
antibodies from two different patients in the 049766 outbreak. Blots in panel C are reacted with anti-108-kDa protein antibodies, and those in panel D are reacted with
anti-116-kDa protein antibodies. Lower-molecular-weight bands in all lanes most likely represent breakdown products of the high-molecular-weight species, since they
are generally absent in blots lacking reactivity in the region from 108 to 116 kDa. MW, molecular weight markers.
FIG. 4. Inhibition of enterotoxicity by antibodies against the peak I fraction.
Twenty-five-microgram aliquots of peak I proteins were preincubated for 20 min
with rabbit serum directed against the identical fraction and then added to the
mucosal hemichambers of rat jejunum preparations (n ? 4).
TABLE 2. Jejunal PD and Isc values after addition of the
108- and 116-kDa EAggEC proteins preheated
or preincubated with proteinase Ka
None None 0.923.89
Peak I proteinsNone
aAverage values recorded 30 min after toxin addition (n ? 4).
b25 ?g of partially purified proteins heated at 75°C for 15 min.
c25 ?g of partially purified proteins preincubated with 200 ?g of proteinase K
per ml for 1 h at 37°C.
VOL. 66, 1998 108-kDa EAggEC ENTEROTOXIN 3151
049766 precipitated supernatant preparations were enriched
by a second precipitation with 1.75 M K2HPO4(Fig. 1, lane B)
and then were separated by DEAE-cellulose chromatography
(lane D). The first peak obtained from the DEAE-cellulose
column (hereafter designated peak I) produced a highly en-
riched fraction containing both the 108- and 116-kDa EAggEC
The peak I supernatant fraction induced increases in rat
jejunal PD and Isc, and affected R, similarly to the crude
049766 precipitates. The effect of peak I proteins on Isc values
was dose dependent (1.5 ?g of protein induced mean increases
in Isc of 2.07 ?A/cm2, while 25 ?g induced mean Isc rises of
13.06 ?A/cm2), starting 20 min after the inoculation (Table 1).
In addition, the same mass of protein (25 ?g) from the peak I
fraction induced a greater increase of PD and Isc than the
crude precipitate (0.71 mV and 14.32 ?A/cm2[peak I] versus
0.44 mV and 10.6 ?A/cm2[crude]). These data strongly suggest
that the 108- and/or the 116-kDa proteins exhibit dose-depen-
dent enterotoxic properties. Interestingly, however, the crude
precipitate produced a significantly greater change in resis-
tance (?R ? 43.2 ?cm2versus 25.06 ?cm2), suggesting that
another factor(s) may also contribute to mucosal damage.
Heat-treated peak I proteins (75°C for 15 min) lost entero-
toxic activity (Table 2). Preincubation with proteinase K also
inhibited the effects of peak I proteins on jejunal PD and Isc
(Table 2). These data are consistent with the presence of a
high-molecular-weight heat-labile protein enterotoxin.
Association of enterotoxic activity with the 108-kDa protein.
The 108- and 116-kDa proteins were found to be immuno-
genic. Serum samples from children with diarrhea due to strain
049766 in the Mexican outbreak, reacted against the superna-
tant of the same strain (Fig. 3A, lane a) by Western immuno-
blotting, recognized either both the 108- and 116-kDa proteins
(Fig. 3B, lane e) or only the 108-kDa species (Fig. 3B, lane f).
We took advantage of the immunogenicity of these proteins
to identify the toxic species. Rabbits immunized with peak I
proteins from strain 049766 produced antibodies against both
the 108- and 116-kDa proteins (Fig. 3B, lane a). Monospecific
polyclonal antibodies against either the 108-kDa (Fig. 3C, lane
a) or 116-kDa (Fig. 3D, lane a) protein were prepared by
excising the proteins from polyacrylamide gels and injecting
the proteins into different rabbits. Each of these antibody prep-
arations was then tested for the ability to inhibit the entero-
toxicity of fractionated EAggEC supernatants in the Ussing
chamber. As expected, anti-peak I antibodies neutralized PD,
Isc, and R changes in Ussing chambers (Fig. 4). No rises in Isc
or decreases in R were detected (Fig. 4A and C). Preincuba-
tion of the peak I fraction with monospecific antibodies against
the 108-kDa but not against the 116-kDa protein neutralized
the effects of the preparation on jejunal PD and Isc (Fig. 5).
These data suggest that the 108-kDa protein is the enterotoxic
species found in the peak I fraction.
We used our 108- and 116-kDa protein-specific polyclonal
antibodies to screen our collection for strains that might ex-
press only the 108- or 116-kDa protein to further support our
hypothesis that the 108-kDa protein was the active species. By
FIG. 5. Inhibition experiments in Ussing chambers with antibodies against
either 108- or 116-kDa protein. Bars 1 to 3, PD and Isc increments induced by
the peak I fraction of strain 049766 alone or after preincubation with monospe-
cific antibodies against either the 108- or 116-kDa protein (n ? 7). Bar 4
represents the rises induced by strain 065126, which lacks the 108-kDa species.
FIG. 6. Enterotoxic activity of ?100-kDa fractionated supernatants contain-
ing the 108-kDa protein. One-hundred micrograms of concentrated supernatant
protein was added to the mucosal hemichambers of rat jejunum preparations
(n ? 4) (see text).
3152 NAVARRO-GARCI´A ET AL.INFECT. IMMUN.
Western immunoblotting strain 065126 was found to express
the 116-kDa (Fig. 3D, lane b) but not the 108-kDa (Fig. 3C,
lane b) protein. As predicted, the ?100-kDa fraction of 065126
(Fig. 3A, lane b) did not induce changes in jejunal PD and Isc
(Fig. 5) and was not significantly different from the preparation
treated with LB medium (P ? 0.1).
Localization of the gene encoding the 108-kDa enterotoxin.
Genetic analyses in our laboratories has focused on EAggEC
strain 042 (7, 24). We decided to use these data to localize the
108-kDa toxin and to substantiate its enterotoxic effects. Con-
centrated supernatants of strain 042 were found to contain the
108- and 116-kDa proteins, detected by SDS-PAGE (Fig. 3A,
lane c) and by immunoblotting with antibodies against 108-
and 116-kDa proteins (Fig. 3B, lane c). As expected from
previous experiments, these concentrated supernatants also
induced increases of PD and Isc (Fig. 6); however, strain 042
cured of its 65-MDa virulence plasmid (pAA2) was found to be
lacking the 108-kDa protein, and the fractionated supernatant
of plasmid-cured 042 had no effect on jejunal preparations
mounted in the Ussing chamber (Fig. 6). We next tested a
series of clones derived from plasmid pAA2 and found that
HB101(pJPN201), harboring a 13-kb insert which flanks the
previously described AAF/II genes (7), expressed the 108-kDa
protein by SDS-PAGE (Fig. 3A, lane d) and by Western blot-
ting (Fig. 3B and C, lanes d). The 116-kDa protein was not
encoded by pJPN201 (Fig. 3D, lane d). Again, as expected,
concentrated fractionated supernatant of HB101(pJPN201) in-
duced rises in jejunal PD and Isc in Ussing chambers (Fig. 6);
the rises were neutralized by anti-108-kDa protein antibodies.
These data confirm that the 108-kDa protein is indeed an
Histopathologic examination of rat mucosal tissue. Since
the 108-kDa protein induced a decrease in electrical resis-
tance, histopathologic analysis of full-thickness rat jejunal tis-
sue was performed by light microscopy after Ussing chamber
experiments. Control-treated rat jejunal sections appeared nor-
mal, with intact mucosa and minimal mucus secretion (Fig.
7A). However, specimens treated with the 108-kDa toxin de-
rived either from 049766 or from 042 [in HB101(pJPN201)]
demonstrated identical histopathologic abnormalities (Fig. 7B).
The mucosal surface of toxin-treated specimens was covered
with a thick mucus blanket. The epithelial layer demonstrated
coagulation necrosis, with exfoliation of epithelial cells and
occasional karyorrhexis of nuclei. Beneath the epithelium were
observed increased numbers of mononuclear cells, and eosin-
ophils and multifocal crypt abscesses were observed in several
specimens. The submucosa exhibited edema and widening of
the lymphatic channels.
EAggEC is an emerging agent of pediatric diarrhea. Clinical-
ly, the disease presents as watery diarrhea, but the responsible
enterotoxin has not yet been identified with certainty. Data do
not yet exist to support a role for the ST-like toxin EAST1
in EAggEC diarrhea (21), and a 120-kDa protein that cross-
reacted with hemolysin antibodies (1) has not been shown to
have enterotoxic properties. Here we present data suggesting
FIG. 7. Morphologic effects of 108-kDa protein on rat jejunal mucosa. The rat jejunal preparations were removed from Ussing chambers, fixed with 4% formalin,
and embedded in paraffin. The sections were stained with hematoxylin and eosin. (A) Untreated control preparation. (B) Preparation treated with 108-kDa protein
from HB101(pJPN201). Note the mucus blanket with cell debris on the luminal side (asterisk), damage of the epithelial layer (arrowhead), and crypt abscesses (arrow)
in the treated section.
VOL. 66, 1998 108-kDa EAggEC ENTEROTOXIN3153
that a 108-kDa protein secreted by EAggEC strains is a heat- Download full-text
labile enterotoxin. This protein is recognized by sera from pa-
tients in an outbreak of EAggEC diarrhea.
The following data suggest that the 108-kDa protein is an
enterotoxin: (i) fractions containing both the 108-kDa protein
and a distinct 116-kDa protein produce rises in Isc, whereas a
fraction from a strain producing only the 116-kDa protein does
not; (ii) polyclonal antibodies raised against the 108-kDa pro-
tein abolish enterotoxic activity in a dose-related fashion,
whereas anti-116-kDa protein antibodies have no effect; (iii) a
108-kDa protein-encoding subclone from the pAA plasmid
induces increases in Isc; a pAA-cured EAggEC strain does not.
The 108-kDa toxin appears to induce not only enterotoxic
effects but also tissue damage, inflammation, and mucus secre-
tion; these effects correlated with a fall in R value. These data
are consistent with other reports that EAggEC strains elabo-
rate one or more cytotoxins and induce damage to the intes-
tinal mucosa (10, 16, 24).
Thus, our data indicate that at least some EAggEC strains
secrete a high-molecular-mass (ca. 108-kDa) protein which is
encoded on the pAA virulence plasmid and has enterotoxic
and perhaps cytotoxic activity on intestinal preparations. The
enterotoxic effects were characterized by an increase of Isc and
PD and a decrease in R, indicating induction of a net secretory
state and damage to epithelial cells and/or their cellular junc-
tions. This enterotoxin is immunogenic, as antibodies against
the 108-kDa protein can be found in sera from children with
EAggEC infection. The 108-kDa enterotoxin could play an
important role in the diarrhea produced by EAggEC.
Our data allow us to hypothesize a model of EAggEC in-
fection in which initial adherence is mediated by AAF fim-
briae, followed by the induction of a net secretory state in-
duced by the 108-kDa enterotoxin and also perhaps EAST1.
This may be followed by the development of cytotoxicity on the
mucosa also induced by the 108-kDa toxin or by an as yet
unidentified factor. Studies to identify these factors are ongo-
This work was supported by grant DGAPA IN-208493 from Uni-
versidad Auto ´noma de Me ´xico and by Public Health Service grant
AI33096 and TW00499 (from the Fogarty Center) to J.P.N.
We thank Klara Margaretten for excellent technical help and Ales-
sio Fasano for use of Ussing chambers at the Center for Vaccine
Development and for assistance in data analysis.
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Editor: J. T. Barbieri
3154NAVARRO-GARCI´A ET AL.INFECT. IMMUN.