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.
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Editor: S. R. Blanke
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