INFECTION AND IMMUNITY, Mar. 2011, p. 1329–1337
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 3
Shiga Toxin Subtypes Display Dramatic Differences in Potency?
Cynthia A. Fuller,1Christine A. Pellino,1Michael J. Flagler,2
Jane E. Strasser,3,4and Alison A. Weiss1*
University of Cincinnati, Department of Molecular Genetics, Biochemistry and Microbiology,1Procter and
Gamble,2University of Cincinnati, Office of Research Compliance and Regulatory Affairs,3and
Cincinnati Children’s Hospital Medical Center, Division of Infectious Diseases,4Cincinnati, Ohio
Received 5 November 2010/Returned for modification 1 December 2010/Accepted 23 December 2010
Purified Shiga toxin (Stx) alone is capable of producing systemic complications, including hemolytic-uremic
syndrome (HUS), in animal models of disease. Stx includes two major antigenic forms (Stx1 and Stx2), with
minor variants of Stx2 (Stx2a to -h). Stx2a is more potent than Stx1. Epidemiologic studies suggest that Stx2
subtypes also differ in potency, but these differences have not been well documented for purified toxin. The
relative potencies of five purified Stx2 subtypes, Stx2a, Stx2b, Stx2c, Stx2d, and activated (elastase-cleaved)
Stx2d, were studied in vitro by examining protein synthesis inhibition using Vero monkey kidney cells and
inhibition of metabolic activity (reduction of resazurin to fluorescent resorufin) using primary human renal
proximal tubule epithelial cells (RPTECs). In both RPTECs and Vero cells, Stx2a, Stx2d, and elastase-cleaved
Stx2d were at least 25 times more potent than Stx2b and Stx2c. In vivo potency in mice was also assessed. Stx2b
and Stx2c had potencies similar to that of Stx1, while Stx2a, Stx2d, and elastase-cleaved Stx2d were 40 to 400
times more potent than Stx1.
Shiga toxin (Stx)-producing Escherichia coli (STEC)
causes approximately 110,000 cases of food-borne illness each
year in the United States (http://www.cdc.gov/nczved/divisions
/dfbmd/diseases/ecoli_o157h7/#who), and these cases range in
severity from mild diarrhea to hemorrhagic colitis. Approxi-
mately 10% of those infected develop life-threatening sequelae
(3, 48, 52), including hemolytic-uremic syndrome (HUS) (38)
and neurological complications (13). This disease dispropor-
tionately affects children under 5 years of age and the elderly
(as mentioned on the above-cited CDC URL).
Stx, the primary virulence factor of STEC, is an AB5toxin
(39). The B pentamer targets cells expressing the glycolipid
globotriaosylceramide (Gb3) (25) and is responsible for deliv-
ery of the A subunit to the cytoplasm. In the cytoplasm, the
enzymatically active A subunit inhibits protein synthesis (39)
by cleaving the N-glycosidic bond of adenine 4324 in 28S
rRNA, preventing tRNA binding (11). Stx includes two major
antigenic forms (Stx1 and Stx2) (54), which share approxi-
mately 60% amino acid identity. Epidemiological studies sug-
gest that Stx2 is more often associated with severe disease and
development of HUS than Stx1 (2, 12, 17, 24, 40, 41). Studies
in primates have shown that administration of Stx2 alone can
produce the symptoms of HUS, while administration of Stx1 at
the same dose does not cause HUS (50, 53). In mouse models,
Stx2 is 100 times more potent than Stx1 (56).
Toxin variants or subtypes share significant amino acid iden-
tity with either Stx1 or Stx2. Stx2 subtypes are also associated
with different clinical outcomes (17, 33, 44, 59). A recent pro-
posal to clarify Stx nomenclature has been published online
(15), and it suggests using classification based on types (Stx1 or
Stx2), followed by subtypes based on nucleotide and amino
acid sequence similarity (e.g., Stx2a to Stx2g), to classify toxins.
Stx homology trees based on DNA relatedness have clearly
established a close relationship among Stx2a, Stx2c, and Stx2d
and have shown that Stx2b and Stx2e to Stx2g are less closely
related (15). In this study, we use the nomenclature proposed
in 2009 (15) and refer to the prototype Stx2 variant from strain
EDL933 as Stx2a. It is important to note that there is consid-
erable confusion in the historic literature, especially for the
Stx2c and Stx2d designations, and some GenBank entries are
clearly misidentified according to the new typing scheme.
Stx2 subtypes may display only a few amino acid changes
(Fig. 1A and B), but these differences appear to influence
disease outcome (15). Strains producing subtype Stx2a, Stx2c,
or Stx2d are often associated with development of hemor-
rhagic colitis (HC) and HUS (2, 24, 40, 41). Stx2a, Stx2c, and
Stx2d display subtle differences in receptor preference (27),
which may influence potency. Strains that produce other (more
distantly related) subtypes (Stx2b and Stx2e) are less fre-
quently associated with human disease. To date, Stx2e has
been associated with potentially fatal edema disease in neona-
tal piglets (32). The molecular basis for the difference in po-
tency is most clear for the Stx2e subtype. Compared to that of
Stx2a, the B subunit of Stx2e has nine amino acid changes and
lacks the two C-terminal amino acids. These changes alter
receptor binding preference: Stx2e binds to the glycolipid glo-
botetraosylceramide (Gb4) instead of Gb3, which is preferred
by Stx1 and most other Stx2 subtypes (57). Interestingly, alter-
ation of only two amino acids in Stx2e (Q64E/K66Q) restored
the preference for Gb3 (30). While epidemiologic data suggest
that particular Stx2 subtypes are more strongly associated with
severe disease, no studies to date have confirmed this with
purified toxins in the absence of confounding factors, such as
differences in strain background, other virulence factors, and
Receptor recognition is primarily mediated by the B penta-
* Corresponding author. Mailing address: Molecular Genetics, Bio-
chemistry, and Microbiology, Room 3109, 231 Albert Sabin Way, ML
524, University of Cincinnati, Cincinnati, OH 45267-0524. Phone:
(513) 558-2820. Fax: (513) 558-8474. E-mail: firstname.lastname@example.org.
?Published ahead of print on 3 January 2011.
mer; however, the A subunit has some role in binding. The C
terminus of the A subunit of Stx2a extends through the pore
formed by the B pentamer (Fig. 1C, in red) and could occlude
binding to a region identified as site 3 in Stx1 (30). Indirect
evidence for a role of site 3 in Stx2 binding came from char-
acterization of Stx2d. The Stx2d subtype has been classified on
the basis of activation by elastase (35), as elastase has been
shown to remove two amino acids (GE) from the C terminus of
the A subunit (Fig. 1A). Elastase-treated culture supernatant
from strains expressing activable Stx2d shows increased Vero
cell cytotoxicity (35), and the increased potency of strains ex-
pressing Stx2d in mouse models of infection (35) and human
disease (1) is thought to be due to toxin activation by intestinal
elastase. The biochemical basis for increased cytotoxicity re-
mains unknown, but it has been hypothesized that removal of
the C-terminal amino acids of the A subunit may make recep-
tor site 3 more accessible.
In this study, the potencies of purified Stx1 and Stx2a to
Stx2d subtypes were examined in vitro using two different pa-
rameters, protein synthesis inhibition and cellular metabolic
toxicity, and in vivo by examining toxicity to mice.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The sources of the strains and puri-
fied toxins used in this study are summarized in Table 1. Stx was purified from
cultures grown in Mueller-Hinton broth (cation stabilized; Difco). L broth or L
agar containing 100 ?g/ml ampicillin and 30 ?g/ml chloramphenicol, as appro-
priate, was used for routine propagation. Tissue culture-grade phosphate-buff-
ered saline (PBS) was obtained from Mediatech (Manassas, VA).
DNA sequencing, analysis, and protein modeling. Coding sequences obtained
from published databases are listed in Table 1. To determine the sequences of
the stx2dA and B subunit genes present in E. coli strain 3024-94 (7), total DNA
was extracted and sequences corresponding to the stxA2dand stxB2dsubunits
were amplified separately by PCR using the primers indicated in Table 1. Am-
plified DNA from three independent experiments was digested with NdeI and
BamHI, cloned into pET21b, and transformed into E. coli DH5?. Plasmid inserts
were sequenced in the forward and reverse directions using T7 forward and T7
terminator primers (Table 1). Identical sequences were obtained for all three
trials. Sequence alignments were made using the NCBI BLAST program (Fig.
1A and B). To generate the predicted molecular structures for Stx2b, Stx2c,
Stx2d, and elastase-cleaved Stx2d (Stx2d?GE; see below), in silico mutagenesis
was performed in PYMOL (DeLano Scientific, Palo Alto, CA) using the coor-
dinates of the Stx2a crystal structure (1R4P) (Fig. 1C).
Stx purification. Stx is encoded as a late phage gene; to improve yields, toxin
was purified from cultures treated with ciprofloxacin to induce the phage lytic
cycle. Overnight cultures were diluted 1/100 in fresh Mueller-Hinton broth and
grown to an optical density at 600 nm of approximately 0.5. Ciprofloxacin (10
ng/ml) was added, and the cultures were incubated overnight at 37°C with
shaking. Cells were removed by centrifugation, and supernatants were filter
sterilized and subjected to ammonium sulfate precipitation. Stx was purified from
the 40 to 80% ammonium sulfate fraction by using AffiGel Blue chromatography,
anion-exchange (Q-Sepharose or UnoQ) chromatography, and size exclusion
(Superdex 75) chromatography. Protein was quantified by using the bicincho-
ninic acid (BCA) assay (Pierce). Only two bands corresponding to the A and B
subunits were visualized by Coomassie staining when 1 ?g of protein was re-
FIG. 1. Sequence alignments and structural models of Stx2 subtypes. Stx2 sequences were aligned using BLASTP (NCBI/BLAST), with periods
indicating identity and dashes indicating absent amino acids. Numbering starts with the first amino acid of the mature peptide; numbers correspond
to bold amino acids. (A) Only the C-terminal 65 amino acids, corresponding to the region of greatest variability, of the 297-amino-acid A subunit
are shown. (B) Alignment of the entire B subunit. (C) The mutagenesis function of PYMOL was used to substitute amino acids of the subtypes
into the Stx2a crystal structure (1R4P). The structures are oriented to display the receptor binding face and are color coded as follows: blue, amino
acids of the Stx2a B pentamer predicted to mediate receptor binding, based on the crystal structure of Stx2e subtype GT3 bound to Gb3 (31); gray,
amino acids not thought to participate in binding; yellow, amino acid polymorphisms in the B subunit of Stx2b to Stx2d; red, the C terminus of
the A subunit; green, amino acid polymorphisms in the A subunit of Stx2b and Stx2d. Red boxes indicate the toxin amino acid variants purified
for this study; the B subunits of Stx2c used in the study and the Stx2c type are identical.
1330 FULLER ET AL.INFECT. IMMUN.
solved on 8 to 16% polyacrylamide gels (Lonza). Lipopolysaccharide (LPS)
content was determined using the limulus amoebocyte lysate (LAL) assay
(Lonza); it was below the limit of detection for Stx2b and Stx2d?GE and less
than 0.02 ng LPS per ?g toxin for Stx2c, Stx2d, Stx1 with the N32S mutation (Stx1
N32S), and Stx1 L41Q. Stx1 and Stx2a were received from the Biodefense and
Emerging Infectious Diseases Research Resources Repository (BEI, Manassas,
VA) and were not tested for endotoxin levels.
Generation and characterization of elastase-activated Stx2d. Stx2d (100 ?g)
was incubated with 10 ?g purified mouse pancreatic elastase (Elastin Products
Company, Owensville, MO) for 30 min at 37°C, followed by the addition of 20 ?g
(5-fold excess) of elastatinal (Sigma, St. Louis, MO) to inhibit elastase activity.
Digested proteins were purified by ion-exchange chromatography (UnoQ), and
two prominent peaks were obtained, corresponding to elastase-digested and
undigested Stx2d. Elastase-treated and control fractions were digested with tryp-
sin (10 ?g Stx2d incubated with 1 ?g trypsin) for 30 min at 37°C to separate the
A1 fragment from the A2 fragment. Stx2d samples were reduced with 200 mM
dithiothreitol, heated at 45°C for 45 min, and analyzed by liquid chromatogra-
phy-mass spectrometry (LC-MS) at the University of Cincinnati Mass Spectros-
copy Core. Removal of the terminal GE from elastase-cleaved Stx2d was con-
firmed by LC-MS (Fig. 2). The A2 fragment of intact Stx2d had a predicted mass
of 5,227 Da (Fig. 2A). Removal of the C-terminal amino acids by elastase is
predicted to generate a 5,040-Da fragment, in agreement with the LC-MS frag-
ment corresponding to 5,042 Da (Fig. 2B). The cleaved Stx2d molecule is re-
ferred to as Stx2d?GE.
Protein synthesis inhibition. Stx-mediated inhibition of protein synthesis was
assessed using Luc2P Vero cells expressing destabilized luciferase as previously
described (34). Briefly, serial dilutions of Stx were added to luminom-
eter-compatible white, tissue culture-treated, Falcon 96-well microtiter plates
(Becton Dickinson, Franklin Lakes, NJ). Luciferase-expressing Vero cells (104)
were added to the wells and incubated with toxin for 4 h at 37°C with 5% CO2.
Cells attach rapidly to the wells even in the presence of toxin. After 4 h, cells were
washed three times with PBS, 25 ?l Superlite luciferase substrate (Bioassay
Systems, Hayward, CA) was added, and light was measured using a Luminoskan
Ascent (Thermo Labsystems, Helsinki, Finland). Luciferase-expressing Vero
cells incubated without toxin were used as the negative control to determine
maximum light production. The effective dose for causing a 50% reduction in
protein synthesis (ED50) was calculated from the dose-response curve by using
the two points above and below the midpoint.
Toxicity to human renal cells. Human RPTECs from a 35-year-old male
(7F4110), and a 7-year-old male (8F3151) were obtained from Clonetics (Lonza,
Walkersville, MD). In initial studies, the cells were propagated by using Clonetics
REGM renal epithelial-cell basal medium in the presence of proprietary growth
factors, cytokines, and supplements as recommended by the manufacturer. How-
TABLE 1. Sources of Stx-producing strains or purified toxins and primers used for sequencing
Toxin or primer Strain Source and/or reference
Protein accession no. (NCBI)
A subunitB subunit
BEI (purified toxin)b
BEI (purified toxin)b
Statens Serum Institut (45)
Sequence comparison only (28)
Statens Serum Institut (44)
Alison O’Brien (55)
Alison O’Brien (7)
aSequence of wild-type Stx1 toxin without the indicated mutation.
bThe following reagents were obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH: recombinant Stx from
E. coli (NR-857) and recombinant Stx type 2 from E. coli (NR-4478).
FIG. 2. Reconstructed mass spectra of Stx2d. Stx2d or elastase-
cleaved Stx2d was treated with trypsin to generate the N-terminal A1
and C-terminal A2 fragments, and the A2 fragments were analyzed by
LC-MS. (A) Trypsin-treated Stx2d control full-length A2 (5,227.4 Da)
is seen as the major peak. (B) Elastase-treated Stx2d. The major
fragment is smaller (5,042.2 Da), corresponding to A2 minus the two
terminal amino acids (GE).
VOL. 79, 2011DIFFERENT POTENCIES OF Stx SUBTYPES1331
ever, RenalLife complete medium (Lifeline Cell Technology, Walkersville, MD)
resulted in better growth and was used for all other studies. Cells were harvested
using TrypKit (Lifeline Cell Technology, Walkersville, MD) according to the
manufacturer’s recommendations. Briefly, ampoules containing 500,000 cells
were thawed, and cells were plated at a density of 2,200 cells per cm2in three
75-cm2tissue culture-treated flasks with vented lids (Becton Dickinson, Franklin
Lakes, NJ). Medium was replaced 24 h after initial plating and every 48 h
thereafter. Division numbers were calculated based on cellular yields. Stx was
serially diluted in 10 ?l of tissue culture-grade PBS and added to sterile, clear,
flat-bottom, tissue culture-treated 96-well microtiter plates (Falcon; Corning,
Lowell, MA). RPTECs (in 100 ?l of medium) were plated at 5 ? 103cells per
well, and the plates were incubated with toxin at 37°C in 5% CO2. After 42 h, the
medium was removed and replaced with 100 ?l of fresh medium containing 10%
(vol/vol) alamarBlue (resazurin; Trek Diagnostic Systems, Cleveland, OH); this
was incubated for 4 h in the presence of alamarBlue to allow reduction of
resazurin to the fluorescent compound resorufin by mitochondrial electron trans-
port. To quantify fluorescence, the medium was transferred into untreated,
black, clear-bottom 96-well plates (Corning, Lowell, MA), and the fluorescence
emission at 590 nm was determined using an FLX-800 fluorimeter (BioTek,
Winooski, VT). Dose-response curves were plotted using the percentage of
untreated cells for the y axis and toxin concentration (pg/ml) for the x axis. The
effective dose to inhibit protein synthesis by 50% (ED50) was calculated from the
x intercept of the line connecting points above and below the midpoint.
Flow cytometry to detect apoptosis. RPTECs at division 8 were treated for 12 h
with PBS, with actinomycin D, an inhibitor of transcription (10 ?M), with
cycloheximide, an inhibitor of protein synthesis (100 ?M), or with Stx2a (0.2
ng/ml). Cells were harvested, washed, stained with Alexa Fluor 488 annexin V
and propidium iodide (PI) according to the manufacturer’s recommendations
(Invitrogen, Carlsbad, CA), and analyzed by flow cytometry. Unstained and
stained cells permeabilized with 0.1% NP-40 (double positive) were used as
negative and positive controls, respectively, for calibration of the flow cytometer
(BD FACSCalibur; Becton Dickinson, Franklin Lakes, NJ).
Toxicity to mice. Male CD-1 mice, 13 to 15 g, were obtained from Charles
River Laboratories (Wilmington, MA) and housed in the animal facilities at the
University of Cincinnati according to the protocol approved by the University of
Cincinnati Institutional Animal Care and Use Committee (IACUC). Three days
after arrival, groups of three mice were injected intraperitoneally (i.p.) with 500
?l of purified toxin diluted into sterile, tissue culture-grade PBS. A positive
control, Stx2a or Stx2d, was included in each trial. Animals were weighed daily;
any animals appearing moribund at a time point or losing more than 20% of
initial body weight 72 h postinjection were sacrificed in compliance with IACUC
regulations. The trial was terminated after 5 days. Mouse survival was plotted
against the log of the toxin dose, and the 50% lethal dose (LD50) was calculated
from the x intercept of the line connecting points above and below the midpoint.
Statistical tests. Statistical analysis (for means, standard deviations, and 95%
confidence intervals) for the Luc2P Vero cell and RPTEC ED50studies and
mouse LD50studies was performed on log-transformed values by using Prism5
(GraphPad Software, La Jolla, CA).
Nucleotide sequence accession numbers. The sequences of stxA2dand stxB2d
were deposited into GenBank, with the corresponding submission accession
numbers HQ585061 and HQ585062, respectively.
Models of Stx2 subtypes. Epidemiological evidence suggests
that E. coli strains expressing different subtypes of Stx2 display
differences in virulence, including the likelihood of progression
to HUS (15, 27, 36, 50). Since different subtypes also display
differences in receptor preference (9, 14, 21, 26, 27, 43), we
wanted to determine if any of the amino acid polymorphisms
map to the Gb3 binding sites in the Stx2 B subunit. The struc-
ture of Stx2a bound to Gb3 has not been determined; however,
the structure of a highly homologous form, Stx2e (GT3), with
bound Gb3 has been determined (31). While Stx2e binds to
Gb4, the mutant, GT3, like Stx2a, binds to Gb3. Since 16 of the
18 amino acids that make contact with Gb3 are identical be-
tween GT3 and Stx2a, this crystal structure is useful for pre-
dicting the Gb3 binding sites on Stx2a.
The structures of the Stx2b, Stx2c, Stx2d, and elastase-
cleaved Stx2d toxins were modeled onto the crystal structure of
Stx2a (1R4P) by using PYMOL software (Fig. 1C), and pre-
dicted Gb3 binding sites were identified on each (they are
shown in blue in Fig. 1C). The Stx2 subtypes share extensive
amino acid homology (Fig. 1A and B); however, several of the
amino acid polymorphisms (Fig. 1C, in yellow) map to, or near,
the predicted receptor binding sites, which suggests that they
could influence binding preferences.
Inhibition of protein synthesis by purified Stx2 subtypes.
Vero cells have been reported to be more sensitive to Stx1 than
Stx2a in assays which assess cellular viability 3 days after treat-
ment (54). In a previous study (27), we assessed the abilities of
Stx1, Stx2a, Stx2c, and Stx2d to mediate protein synthesis in-
hibition by using Vero cells which had been modified to ex-
press a destabilized form of luciferase (34); Stx1 was found to
be more potent than Stx2a, while Stx2c was significantly less
potent than either Stx1 or Stx2a (Table 2) (27). We obtained
almost identical results when these experiments were repeated
in the current study. In the current study, the abilities of Stx2b
and Stx2d?GE to inhibit protein synthesis were also assessed
(Table 2). The potency of Stx2b was similar to those of Stx2a
and Stx2d. The activated form of Stx2d, Stx2d?GE, was ap-
proximately 5 times more potent than Stx2d, but this difference
did not achieve statistical significance.
Toxicity to primary human kidney cells. RPTECs were
treated with purified toxin for 42 h, and mitochondrial activity
was assessed by monitoring reduction of resazurin to the flu-
orescent compound resorufin. In initial studies, RPTECs from
a 35-year-old male were cultured in REGM renal epithelial-
cell basal medium from Clonetics. A large number of cells
from the ampoule failed to adhere, and it took several days for
the plate to become confluent. The cells displayed an esti-
mated doubling time of 36 h. Sensitivity to three subtypes,
Stx2a, Stx2c, and Stx2d, was dependent on the number of cell
divisions after initial plating (Fig. 3A), with cells at division 16
being approximately 100-fold less sensitive than cells at divi-
TABLE 2. ED50s of Stx variants for Vero cells and RPTECs and
LD50s for mice
ED50(pg/ml) (95% CI)a
Vero cells RPTECs
46,700 (24,700–88,400)c32,000 (7,790–131,000)
aED50values were calculated by determining the x intercept of the line
connecting the points above and below the midpoint on plots of dose-response
curves. The 95% confidence intervals were determined using log-transformed
ED50values in Prism5 (GraphPad Software, La Jolla, CA), and values are
expressed as the ED50value with the 95% confidence interval values in paren-
theses. *, significant difference (nonoverlapping confidence interval) between
Vero cells and RPTECs.
bLD50s were calculated by determining the x intercept of the line connecting
the points above and below the midpoint of plots of survival data at 120 h
postinjection (i.p.) of Stx.
cValues taken from published studies (27). Similar values were obtained when
these experiments were repeated for the current study.
1332FULLER ET AL.INFECT. IMMUN.
sion 12. Giemsa staining of RPTECs revealed a more diverse
appearance at later cell divisions. After 16 passages, cells sur-
viving Stx treatment were larger than the untreated cells, sug-
gesting that the large cells are resistant to Stx.
To improve initial recovery and cellular growth, RPTECs
were cultured in RenalLife complete medium from Lifeline.
Adhesion of RPTECs from cryovials incubated in the Lifeline
medium was remarkably increased compared to that of cells in
Clonetics medium, and the doubling time was approximately
24 h. At division 10, cells grown in the Clonetics medium were
much more sensitive to Stx2a and Stx2d than to Stx2c (Fig. 3B).
Children are more likely to develop HUS than adults (18,
22). RPTECs from the youngest donor available, a 7-year-old
male, were also assessed for sensitivity to Stx2. When cells were
cultured under identical conditions, ED50values for cells from
the 7-year-old male were not significantly different from those
for the corresponding cells of the 35-year-old male, except
for the Stx2a ED50s (P ? 0.14; not shown). Considerable
differences in potency were observed for the different sub-
types (Fig. 3B).
The ED50values were calculated and are shown in Table 2.
The activated form of Stx2d, Stx2?GE (ED50? 1 pg/ml),
was the most potent form, followed by Stx2d (ED50? 4 pg/ml)
and then Stx2a (ED50? 28 pg/ml). Stx2?GE was significantly
more potent than Stx2a but not Stx2d. The cells were much less
sensitive to Stx2b (ED50? 718 pg/ml) and Stx2c (ED50?
32,000 pg/ml). Stx2b and Stx2c were significantly less potent
than Stx2a, Stx2d, and Stx2?GE. Unlike the Vero cells,
RPTECs displayed equivalent susceptibilities to Stx1 and Stx2.
The ED50s of Stx2a, Stx2d, and Stx2?GE for the human
RPTECs were significantly lower than those for the Vero
cells, while differences between Stx1, Stx2b, and Stx2c ED50s
were not significantly different.
Two mutants of Stx1 were also tested. Stx1 N32S was gen-
erated to replace the B subunit amino acid at position 32 with
the corresponding amino acid from Stx2a, which causes the
mutant to display Stx2a-like binding preferences for synthetic
glycans (14). Stx1 L41Q was generated to replace the B subunit
amino acid at position 41 with the corresponding amino acid
from Stx2a, a change which decreases the B pentamer stability
(5). The potencies of the mutants were not statistically differ-
ent from that of the wild-type Stx1 (Fig. 3D).
Stx2a does not promote apoptosis in RPTECs. Previous
studies reported that Stx2a induces apoptosis in various cell
lines (4, 17, 20) and serum-starved RPTECs (6, 51) obtained
from renal cell carcinomas. We examined the ability of Stx2a to
induce apoptosis in RPTECs grown in Lifeline medium con-
taining 0.5% serum. Cellular viability was assessed using pro-
pidium iodide, which is excluded from cells with an intact cell
membrane but stains the nuclei when the cytoplasmic mem-
brane is compromised. Apoptosis was assessed using annexin
V. Annexin V binds to phosphatidylserine, which is translo-
cated from the cytoplasmic face of the membrane to the cell
surface during the early stages of apoptosis.
FIG. 3. Stx-mediated inhibition of metabolic activity of primary human kidney cells. RPTECs from two donors were grown in the indicated
medium and treated with dilutions of purified Stx as indicated. After 48 h, medium was replaced with the medium containing 10% (vol/vol)
alamarBlue, cells were incubated for an additional 4 h, and fluorescence of reduced alamarBlue was measured. The graphs depict toxin-treated
cells standardized to percent metabolic activity of untreated control cells. (A) RPTECs, from a 35-year-old male, that were grown in Clonetics
medium. Metabolic activity was assessed at division 12 or 16 after 2 days’ incubation with toxin. (B) RPTECs, from a 35-year-old male, that were
grown in Lifeline medium were treated with Stx2 at division 8 and assessed for metabolic activity at division 10. (C) RPTECs, from a 7-year-old
male, that were grown in Lifeline medium were treated with Stx2 at division 8 and assessed for metabolic activity at division 10. (D) RPTECs, from
a 7-year-old male, that were grown in Lifeline medium were treated with Stx1 and mutants at division 8 and assessed for metabolic activity at
division 10. Error bars depict standard errors of the means, which were calculated in Prism5 (GraphPad Software, La Jolla, CA).
VOL. 79, 2011 DIFFERENT POTENCIES OF Stx SUBTYPES1333
At 12 h posttreatment, about 5% of the untreated RPTECs
appeared to be dead, as evidenced by elevated staining with
propidium iodide (Fig. 4A, right half), and about 30% of the
cells displayed elevated staining with annexin V (Fig. 4A, up-
per left quadrant). These results are consistent with the obser-
vation that these primary cells have a limited life span in vitro
and evidence that apoptosis is seen even at division 8. Treat-
ment with actinomycin D, an inhibitor of RNA polymerase,
induced apoptosis (Fig. 4B), as evidenced by an increase in
propidium iodide-positive dead cells (from 5% to 10%) and
the annexin V-positive apoptotic cells (from 29% to 43%). In
contrast, treatment with cycloheximide, an inhibitor of protein
synthesis did not cause an increase in either propidium iodide
or annexin V staining, suggesting that it did not promote apop-
tosis (Fig. 4C). Similarly, Stx2a, which also inhibits protein
synthesis, did not cause an increase in either propidium iodide
or annexin V staining (Fig. 4D), suggesting that Stx2a also did
not promote apoptosis in cells obtained from healthy kidneys
grown in the presence of serum. However, a significant pro-
portion of the untreated control cells displayed elevated ex-
pression of annexin V, a marker for early apoptosis, and it is
likely that apoptosis plays a role in limiting the life span of
these primary cells in vitro.
Determination of LD50values in mice. Male CD-1 mice
were injected i.p. with half-log dilutions of purified toxin rang-
ing from 0.3 to 1,000 ng per mouse. Survival results are sum-
marized in Fig. 5. Stx2a, Stx2d, and Stx2d?GE displayed sim-
ilar potencies, with LD50values ranging from 2 to 23 ng (Fig.
5 and Table 2), and were significantly more potent than Stx2b
or Stx2c. All Stx2b-treated mice survived regardless of the
dose, and all mice treated with less than 1,000 ng of Stx2c
survived. The LD50s of Stx2b and Stx2c were similar to those of
Stx1 and the mutants Stx1 N32S and Stx1 L41Q (Fig. 5 and
While administration of purified Stx is sufficient to cause
HUS in primate models of disease (50, 53), the ability to
predict whether intestinal infection will progress to HUS is
severely limited. Both human and microbial factors influence
the outcome of infection due to STEC (Fig. 6). Children and
the elderly are more likely to develop HUS, although the basis
for age-related susceptibility is unclear, and it is likely that
different factors mediate susceptibility in each age group. It is
also clear that host E. coli genetics influence pathogenic po-
tential, and STEC strains which lack intestinal colonization
FIG. 4. Flow cytometry analysis of annexin V binding versus pro-
pidium iodide staining. RPTECs at division 8 were treated for 12 h as
indicated with PBS (control) (A), actinomycin D (inhibition of tran-
scription, 10 ?M) (B), cycloheximide (inhibition of protein synthesis,
100 ?M) (C), or Stx2a (inhibition of protein synthesis) (D). Cells were
stained with annexin V and propidium iodide. Percentages of cells in
each quadrant are indicated.
FIG. 5. Mouse toxicity studies. Male CD-1 mice were injected i.p.
with half-log dilutions of purified Stx ranging from 0.3 to 1,000 ng per
mouse and monitored for 120 h. The LD50values are marked by a bar
and are noted at the bottom of the columns.
1334 FULLER ET AL.INFECT. IMMUN.
factors such as intimin and Tir are not associated with clinical
disease (12, 17). Other microbial factors are likely important
but are less well characterized.
Originally, clinical disease due to STEC was predominantly
associated with E. coli serotype O157:H7. It is clear that, for E.
coli strains with the fairly consistent O157:H7 genetic back-
ground, humans infected with isolates that produce Stx2a are
more likely to develop life-threatening disease than individuals
infected with isolates that produce Stx1 (2, 12, 24, 40, 41).
However, even the pathogenic potentials of Stx2a-producing
O157:H7 strains can differ (8, 36). In an interesting study by
Muniesa et al. (36), Stx2a production levels of O157:H7 iso-
lates from a single outbreak were found to differ, and an
adverse clinical outcome was associated with high-level Stx
production. Further genetic characterization demonstrated
that the low-level toxin producers had two copies of an Stx2-
encoding phage and converted to high-level toxin production
when one phage copy was deleted, suggesting a complex reg-
ulation where one phage could suppress toxin production from
both toxin alleles. Muniesa et al. (36) demonstrated that bac-
terial isolates from the same lineage can display different
pathogenic potentials, and the presence of a toxin gene does
not mean it will be expressed. Predicting the pathogenic po-
tentials of non-O157:H7 strains is even more complicated, and
it is difficult to determine whether pathogenic potential is most
influenced by the E. coli genetic background or the Shiga toxin
The goal of this study was to examine a single variable, the
relative potency of purified Stx2 subtypes in vitro by using
primary cells thought to be a target in human disease (renal
proximal tubule cells) and in vivo by using a mouse model.
Interestingly, human RPTECs displayed age-dependent sus-
ceptibility to Stx2a; kidney cells from a 7-year-old were more
susceptible than those from a 35-year-old (Fig. 3). However,
experiments with primary cells must be carefully controlled, as
indicated by the variability of RPTEC susceptibility depending
on culture conditions (growth medium and number of dou-
blings in vitro, etc.).
Stx2a did not mediate death through an apoptotic process
in our studies using cells harvested from healthy kidneys and
grown in the presence of serum (Fig. 4). In contrast, apoptosis
was observed previously when experiments were performed
using transformed cells (4, 17, 20) or primary cells obtained
from renal carcinoma patients (6, 53). The role of apoptosis in
Stx-mediated disease is also unclear. Several studies utilizing
mouse models have observed apoptosis in renal cells following
treatment with Stx (23, 42, 47), while other studies have ob-
served necrosis (10, 29, 37, 46, 58, 60).
The rank order of Stx potency in human RPTECs was the
same as the rank order of toxin-mediated protein synthesis
inhibition using Vero cells (Table 2): Stx2d?GE, intact Stx2d,
and Stx2a have similar potencies, while Stx2b and Stx2c display
greatly reduced potencies. There was a similar rank order for
potency in mice, except that Stx2b appeared to be less potent
than Stx2c (Fig. 6). The relative lack of potency for Stx2c in all
three toxicity assays is puzzling, since Stx2c has been reported
to be associated with HUS (12, 16, 33, 44). However, there is
some confusion in the literature: sequences deposited in
GenBank as Stx2c would be unambiguously classified as Stx2d
using the designations adopted in 2009 (15). Unfortunately,
since strain names are not always linked to clinical outcome,
it is not possible to reevaluate published clinical correlations
using the new standardized subtype designations. Neverthe-
less, newer reports using the current nomenclature have linked
production of Stx2c to HUS (44). Stx2c-producing isolates
associated with HUS could possess an especially potent com-
bination of E. coli virulence factors or produce especially high
levels of toxin. While mice are very sensitive to toxin admin-
istered by intravenous or i.p. injection (a route which bypasses
the intestinal barrier), the mouse intestinal tract lacks the
Shiga toxin receptor, Gb3 (19), and mice are extremely resis-
tant to toxin delivered via intestinal infection (35). The mech-
anism of transfer of Stx from the intestinal tract to the circu-
lation in human disease is poorly understood, but efficient
transfer could be a determinant of potency that would go
unrecognized in these studies. In addition, it is well known that
strains that produce only Stx1 occasionally cause HUS (2, 12,
17, 24, 40, 41), perhaps due to infection of exceptionally sus-
ceptible hosts. However, it is important to note that unlike
Stx1, which is highly toxic to kidney cells, Stx2c is not highly
toxic to kidney cells (Table 2); therefore, it is likely that Stx1
and Stx2c lack potency for different reasons.
Our studies also replicated the in vitro potency versus in vivo
potency paradox associated with Stx1, whereby Stx1 is more
toxic to Vero cells than Stx2a (54) but much less toxic to mice
in vivo (56). In vivo localization studies have shown that Stx1
accumulates in the lungs of mice, while Stx2 does not (49), and
it has been suggested that the lack of potency of Stx1 is due to
binding to toxin-resistant cells in the lungs, thereby protecting
the vulnerable cells in the kidney. This is consistent with our
observation that, in contrast to the case for Vero cells, Stx1 and
Stx2 display equivalent toxicities to RPTECs (human kidney
cells). It is interesting that the more potent toxin forms, Stx2a,
Stx2d, and Stx2?GE, are 16 to 90 times more toxic to RPTECs
than to Vero cells (Table 2). It is possible that this correlation
is true only for cells from the 7-year-old male, since cells from
the 35-year-old male were less susceptible to Stx2a; more de-
tailed studies of the RPTECs may yield insights into age-
While the molecular basis of potency remains unknown, the
potent Stx2d subtype displays the wild-type Stx2a B subunit
receptor binding surface, since the single amino acid difference
between Stx2a and Stx2d does not map to the receptor binding
face (Fig. 1C, note the lack of amino acids in yellow). In
contrast, for the less potent subtypes, one of the two amino
acid polymorphisms of the Stx2c B subunit and three of the five
FIG. 6. Interactions of multiple factors contributing to the severity
of Stx-mediated disease.
VOL. 79, 2011DIFFERENT POTENCIES OF Stx SUBTYPES 1335
polymorphisms in the Stx2b B subunit map to the binding face
(Fig. 1, yellow). The prototype Stx2d variant expresses the
same receptor binding surface as the nonpotent Stx2c subtype
and might be predicted to have reduced potency. A more
detailed understanding of receptor preferences of the Stx2
subtypes is needed.
This work was supported by National Institutes of Health Grants
R01 AI064893 and U01 AI075498 and a grant from the Midwest
Center for Emerging Infectious Diseases to A.A.W.
The following reagents were obtained through the NIH Biodefense
and Emerging Infections Research Resources Repository, NIAID,
NIH: recombinant Stx from E. coli (NR-857) and recombinant Stx type
2 from E. coli (NR-4478).
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Editor: S. R. Blanke
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