MAJOR ARTICLE
Acquisition of stcE, a C1 Esterase Inhibitor–
Specific Metalloprotease, during the Evolution
of Escherichia coli O157:H7
Wyndham W. Lathem,1 Tessa Bergsbaken,1 Sarah E. Witowski,1 Nicole T. Perna,2 and Rodney A. Welch1
Departments of 1Medical Microbiology and Immunology and 2Animal Health and Biomedical Sciences, University of Wisconsin, Madison
Escherichia coli O157:H7 is a source of foodborne illness, causing diarrhea, hemorrhagic colitis, and hemolyticuremic syndrome. E. coli O157:H7 secretes, via the etp type II secretion system, a metalloprotease, StcE, that
specifically cleaves the serpin C1 esterase inhibitor. We determined by hybridization techniques the prevalence
of stcE and etpD, a type II secretion gene, among diarrheagenic E. coli strains. stcE and etpD are ubiquitous
among the O157:H7 serotype and are found in some enteropathogenic E. coli O55:H7 strains but are absent
from other diarrheagenic E. coli. stcE was acquired on a large plasmid early in the evolution of E. coli O157:
H7, before the inheritance of the Shiga toxin prophage. Other plasmidborne virulence factors, such as ehxA,
katP, and espP, were acquired later by the enterohemorrhagic E. coli 1 complex in a stepwise manner. These
data refine the sequential model of E. coli O157:H7 evolution proposed elsewhere.
Shiga toxin (Stx)–producing Escherichia coli (STEC) are
important human pathogens that cause diarrhea, hemorrhagic colitis, and the potentially lethal hemolyticuremic syndrome. STEC are a heterogeneous group of
diarrheagenic E. coli serotypes that carry a diverse collection of genetic elements and virulence factors [1].
Thirty-six distinct stx-positive O:H serotypes have been
isolated from humans [2], which fall into 4 main groups
on the basis of electrophoretic type (ET) [3]. A subset
of these are classified as enterohemorrhagic E. coli
(EHEC), consisting of strains of the serotypes O26:H11
Received 12 September 2002; accepted 18 January 2003; electronically published
4 June 2003.
Presented in part: 101st general meeting of the American Society for Microbiology,
Orlando, Florida, 20–24 May 2001 (abstract B-321).
Financial support: National Institutes of Health (AI20323 and AI51735 to R.A.W.);
US Food and Drug Administration (FD-U-001627–02–1 to N.T.P.); University of Wisconsin School of Medicine; Food Research Institute, University of Wisconsin College of Agriculture and Life Sciences.
Reprints or correspondence: Dr. Rodney A. Welch, Dept. of Medical Microbiology and Immunology, 1300 University Ave., Madison, WI 53706 ([email protected]).
The Journal of Infectious Diseases 2003; 187:1907–14
2003 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2003/18712-0010$15.00
(EHEC 2 complex), O111:NM (EHEC 2 complex), and
O157:H7 (EHEC 1 complex) [4]. The O157:H7 serotype, which is a major cause of infectious gastrointestinal disease in the United States, Europe, and Japan,
is only distantly related to other STEC strains and E.
coli K-12 [4–7]. It is hypothesized to have evolved sequentially from an enteropathogenic E. coli (EPEC)
O55:H7 ancestor (itself distantly related to other EPEC
serotypes [8]) by stepwise changes in phenotypic markers and virulence traits [9]. Central to this hypothesis
is the acquisition of the stx2 gene by E. coli O55:H7,
followed by a divergence into 2 branches, one represented by the loss of b-glucuronidase activity and the
ability to ferment sorbitol and the other by the loss of
motility, leading to the O157:H⫺ serotype [9].
STEC carry large plasmids of various sizes, often
75–100 kb [10–13]. Recent studies have shown these
plasmids to be highly heterogeneous, harboring different collections of genes [13]. Of these, the large plasmid of E. coli O157:H7 strain EDL933 (pO157) is 92
kb and contains 100 open-reading frames [14]. The role
of pO157 in the pathogenesis of E. coli O157:H7 is unclear, because the results of animal studies have been
Acquisition of stcE by E. coli O157:H7
• JID 2003:187 (15 June) • 1907
in conflict [15]. However, genes for multiple putative virulence
factors have been identified on pO157, including the EHEC
hemolysin (ehxCABD) [16, 17], an extracellular serine protease
that cleaves human coagulation factor V (espP) [18], a periplasmic catalase-peroxidase (katP) [19], and a ToxB homologue
from Clostridium difficile that may have lymphostatic activity
[20] and may contribute to epithelial cell adherence [21].
pO157 also carries 13 genes (etpC–etpO) that encode a functional type II secretion apparatus [22, 23].
Our laboratory recently identified an additional potential
virulence factor, StcE, that is encoded on pO157 [23]. StcE is
a metalloprotease that specifically cleaves C1 esterase inhibitor
(C1-INH), a host regulator of multiple proteolytic cascades,
including classical complement, intrinsic coagulation, and contact activation [24]. StcE is secreted via the etp-encoded type
II secretion pathway and is positively regulated by the locus of
enterocyte effacement–encoded regulator, Ler [23, 25].
The highly variable nature of STEC plasmids and the proposed model of sequential E. coli O157:H7 evolution led us to
examine the distribution of stcE among diarrheagenic E. coli
and the point at which these strains may have gained the gene.
Our results demonstrate that stcE is widely distributed in the
EHEC 1 group but is largely absent from other STEC strains.
stcE was acquired on a plasmid early in the evolution of E. coli
O157:H7 and precedes the acquisition of other plasmidborne
virulence factors. In sum, our data refine the model of sequential E. coli O157:H7 evolution proposed by Feng et al. [9].
MATERIALS AND METHODS
Materials, bacterial strains, and culture conditions.
All
chemicals were purchased from Sigma, unless stated otherwise.
Bacterial strains are listed in table 1. Bacteria were grown in
Luria-Bertani (LB) medium or Lennox L broth [33] overnight
at 37C with agitation or on LB medium with antibiotic selection, where appropriate (ampicillin, 100mg/mL, and kanamycin,
50 mg/mL). All strains were stored at ⫺80C in LB medium
with 40% glycerol or in brain-heart infusion broth with 30%
glycerol.
Polymerase chain reaction (PCR). The primers used in
this study are listed in table 2. Genomic DNA was isolated from
bacterial strains with the Wizard Genomic DNA preparation
kit (Promega). Regions of stcE were amplified with the primer
pairs stcE5846/stcE31773 and stcE51/stcE32798, covering the
middle third and entire length of the promoter and the coding
region, respectively. Primers etpD5571 and etpD31632 were
used to amplify an internal 1061-bp fragment of the etpD gene;
primers hlyA1 and hlyA4, to amplify a 1551-bp fragment of
ehxA; primers espP1 and espP2, to amplify a 1830-bp region
of espP; and primers katP1 and katP2, to amplify a 2125-bp
fragment of the katP gene. PCR products were detected by
agarose gel electrophoresis and ethidium bromide staining.
Colony blot analyses. A 927-bp fragment of the stcE gene
was amplified from pO157 by PCR, using the primers stcE5846
and stcE31773. The resulting product was labeled with 32P[dCTP]
Table 1. Bacterial strains used in a study of the distribution of stcE among diarrheagenic
Escherichia coli (DEC).
Strain(s)
Description
Source
Reference
a
EDL933
E. coli O157:H7 strain
A. O’Brien
EDL933cu
EDL933 cured of pO157
A. O’Brien
[26]
[26]
DEC collection
DEC strains
T. Whittamb
[5]
MDH collection
E. coli O157:H7 clinical isolates
MDHc
ECOR37
E. coli ON:H7 isolate
T. Whittam
CDC G5101
E. coli O157:H7 clinical isolate
STEC Center
493/89
E. coli O157:H⫺ clinical isolate
STEC Center
[29]
FDA 413
E. coli O157:NM clinical isolate
[27]
b
[28]
STEC Center
[30]
2937
⫺
E. coli O157:H clinical isolate
STEC Center
[31]
6790
E. coli O157:H⫺ clinical isolate
STEC Center
[31]
431
E. coli O157:H⫺ clinical isolate
STEC Center
[31]
659
E. coli O157:H⫺ clinical isolate
STEC Center
[31]
2576
⫺
E. coli O157:H clinical isolate
STEC Center
[31]
5905
E. coli O157:H7 clinical isolate
STEC Center
[9]
TB156A
E. coli O55:H7 clinical isolate
STEC Center
[32]
TB182A
E. coli O55:H7 clinical isolate
STEC Center
[32]
NOTE.
a
b
c
MDH, Minnesota Department of Health; STEC, Shiga toxin–producing E. coli.
Uniformed Services Medical School, Bethesda, MD.
National Food Safety and Toxicology Center, Michigan State University, East Lansing.
Minneapolis.
1908 • JID 2003:187 (15 June) • Lathem et al.
Table 2. Primers used in a study of the distribution of stcE among diarrheagenic
Escherichia coli.
Name
Sequence
Reference
stcE5 1
5 -TTTACGAAACAGGTGTAAATATGTTATAAA-3
stcE5846
5GAGATTAATCGAATCACTTATCGTC-3
stcE3 1773
5 -CGGTGGAGGAACGGCTATCGA-3
stcE32798
5-TTATTTATATACAACCCTCATTGACCTAGG-3
etpD5571
5CGTCAGGAGGATGTTCAG-3
etpD31632
5-CGACTGCACCTGTTCCTGATTA-3
Present
Present
Present
Present
[22]
[22]
hlyA1
5 -GGTGCAGCAGGAAAAGTTGTAG-3
hlyA4
5-TCTCGCCTGATAGTGTTTGGTA-3
[17]
espP1
5-AAACAGCAGGCACTTGAACG-3
[13]
espP2
5-GGAGTCGTCAGTCAGTAGAT-3
katP1
5 -CTTCCTGTTCTGATTCTTCTGG-3
katP2
5-AACTTATTTCTCGCATCATCC-3
(DuPont NEN), using the Prime-It RmT system (Stratagene).
Bacterial strains were patched onto Magna Lift nylon transfer
membranes (Osmonics) and grown on LB plates overnight at
room temperature. Colonies were lysed by placing the membranes on 3MM Whatman paper soaked in 0.5 M NaOH. Neutralization was performed by placing the membranes on 3MM
Whatman paper soaked in 1 M Tris (pH 7.5) and then on 3MM
Whatman paper soaked in 0.5 M Tris (pH 7.5)/1.25 M NaCl.
The DNA was then cross-linked using UV irradiation. Hybridization was performed at 60C overnight in Church buffer
(0.5 M sodium phosphate [pH 7.2], 7% SDS, 1% bovine serum
albumin, and 1 mM EDTA). The membranes were washed at
60C in 1⫻ sodium citrate buffer (SSC)/0.1% SDS for 15 min
and then in 0.5⫻ SSC/0.1% SDS for 15 min before exposure.
Protein isolation. Bacterial cultures were grown in 50 mL
of Lennox L broth at 37C overnight with aeration. Culture
supernatants were harvested by centrifugation for 15 min at
9000 g and then filtered through a 0.45-mm filter. Supernatants
were precipitated with ammonium sulfate to 75% saturation.
The precipitates were centrifuged for 15 min at 16,000 g, resuspended in 500 mL of AD buffer (20 mM Tris [pH 7.5], 10%
glycerol, 100 mM NaCl, and 0.01% Tween-20), and dialyzed
overnight against 3 changes of AD buffer.
C1-INH proteolysis.
Purified CI-INH (1 mg; Cortex
Biochem) was mixed with ammonium sulfate–precipitated bacterial culture supernatants (200 mL) at room temperature overnight and precipitated with 10% trichloroacetic acid at 4C for
at least 1 h before analysis by immunoblot. No attempt was
made to normalize for protein content before analysis.
Immunoblot analyses. Immunoblot analyses with either
polyclonal anti–C1-INH antibody (1:2000) or polyclonal anti–
StcE-His antibody (1:500) were performed as described elsewhere [23].
Southern blot analyses. Isolation of plasmid DNA species
[17]
[13]
[19]
[19]
present in bacterial strains was performed as described elsewhere [34]. Plasmid preparations were electrophoresed on a
1% agarose gel in Tris-acetate-EDTA. The gel was soaked in
0.23 M HCl for 15 min, 0.5 M NaOH/1.5 M NaCl for 45 min,
and 10⫻ SSC for 15 min before transfer to nylon filters (Amersham). The promoter and coding region of stcE was amplified
by PCR from pO157 with the primers stcE51 and stcE32798.
The resulting 2.8-kb product was labeled with 32P[dCTP] using the Prime-It RmT system, hybridized with the blot, and
washed, as described above.
RESULTS
Prevalence of stcE strains from the diarrheagenic E. coli (DEC)
and Minnesota Department of Health (MDH) collections.
Previous research in our laboratory identified a protease, StcE,
that was present in EHEC strain EDL933 but absent from EPEC
strain E2348/69 [23]. To establish the distribution of StcE among
pathogenic E. coli, we screened the DEC collection (STEC Center,
National Food Safety and Toxicology Center, Michigan State University, East Lansing) with an internal 927-bp probe for the presence of stcE by colony blot analysis. Of 74 DEC strains examined,
13 were positive (table 3). These included DEC 3A–3E (serotype
O157:H7), DEC 4A–4E (serotype O157:H7), and DEC 5A, 5C,
and 5E (serotype O55:H7). All other strains in the DEC collection
failed to hybridize with the stcE probe.
To establish the distribution of stcE among a larger collection
of E. coli O157:H7 isolates, we examined by PCR 72 strains cultured from clinical samples from the upper Midwest. Of 72
O157:H7 isolates tested in the MDH collection (Minneapolis), 61 (85%) were positive for stcE, demonstrating the broad
distribution of stcE among clinical strains of this serotype. We
also established the presence of stcE in several other isolates,
including strains ECOR37 (serotype ON:H7), 5905 (serotype
Acquisition of stcE by E. coli O157:H7 • JID 2003:187 (15 June) • 1909
Table 3. Prevalence of stcE and its associated activity among
strains in the diarrheagenic Escherichia coli (DEC) collection.
Strain
Predominant
serotype
Disease
category
stcE
stcE activity
EDL933
O157:H7
EHEC
+
+
DEC 1A–1E
O55:H6
EPEC
⫺
ND
DEC 2A–2E
O55:H6
EPEC
⫺
ND
DEC 3A–3E
O157:H7
EHEC
+
+
DEC 4A–4E
O157:H7
EHEC
+
+
DEC 5A
O55:H7
EPEC
+
+
DEC 5B
O55:H7
EPEC
⫺
⫺
DEC 5C
O55:H7
EPEC
+
⫺
DEC 5D
O55:H7
EPEC
⫺
⫺
DEC 5E
O55:H7
EPEC
+
+
DEC 6A–6E
O111:H12
EPEC
⫺
ND
DEC 7A–7E
O157:H43
ETEC
⫺
⫺
DEC 8A–8E
O111:H8
EHEC
⫺
ND
DEC 9A–9E
O26:H11
EHEC
⫺
ND
DEC 10A–10E
O26:H11
EHEC
⫺
ND
DEC 11A–11E
O128:H2
EPEC
⫺
ND
DEC 12A–12E
O111:H2
EPEC
⫺
ND
DEC 13A–13E
O128:H7
ETEC
⫺
ND
DEC 14A–14E
O128:H21
EPEC
⫺
ND
DEC 15A–15E
O111:H21
EPEC
⫺
ND
NOTE. Strains were examined by colony blot for the presence of a
927-bp region internal to the coding sequence of stcE. Culture supernatants
from the same strains were also tested for the ability to degrade C1 esterase inhibitor, an activity associated with StcE. Strains are grouped according to electrophoretic type [5], usually 4–5 strains/group. EHEC, enterohemorrhagic E. coli; EPEC, enteropathogenic E. coli; ND, not done; +,
positive/present; ⫺, negative/not present.
O55:H7), CDC G5101 (serotype O157:H7), and TB182A (serotype O55:H7).
Production and activity of StcE. StcE is actively secreted via the etp type II secretion cluster encoded on pO157 in
EDL933 and cleaves C1-INH [23]. To test whether strains carrying the gene for stcE express the protease, we examined these
strains for the secretion and activity of StcE. Although the O157:
H7 strains and 3 of 5 O55:H7 strains in the DEC collection
contain the gene for stcE, it is possible that they do not secrete
an active protease. To test this possibility, the production and
secretion of StcE by these strains were examined. We concentrated culture supernatants from these strains by ammonium
sulfate precipitation and screened for the presence of StcE by
immunoblot analysis, using a polyclonal anti-StcE antibody. All
O157:H7 strains and 2 of 3 O55:H7 strains contained reactive
polypeptides that corresponded to the apparent molecular
weight of StcE from EDL933 (figure 1A). One O55:H7 strain,
DEC 5C, lacked a polypeptide of the appropriate size in the
concentrated culture supernatant.
To test for C1-INH–specific proteolytic activity produced by
1910 • JID 2003:187 (15 June) • Lathem et al.
DEC 3, 4, and 5, we combined concentrated culture supernatants from these strains with purified C1-INH. The mixtures
were incubated overnight and then analyzed by immunoblot
for the presence of full-length C1-INH, using an anti–C1-INH
antibody. Reduction in the amount of full-length C1-INH,
compared with untreated C1-INH, was considered a positive
reaction. The culture supernatants from strains that contained
StcE-reactive polypeptides were able to degrade full-length C1INH to some degree (figure 1B and table 3). As predicted by
the absence of an StcE-reactive polypeptide in the culture supernatant, strain DEC 5C was unable to proteolyze C1-INH.
Association of stcE with plasmid DNA in DEC 5A, 5C, and
5E. In EDL933, the gene for stcE is found immediately 5 to
the etp type II secretion gene cluster on the pO157 plasmid
[14]. To determine the plasmid or chromosomal location of
stcE in DEC 5A, 5C, and 5E, we used the 927-bp internal stcE
fragment as a probe in Southern blot analyses of rapid plasmid
DNA preparations from EDL933, EDL933cu, and the DEC 5
strains. We observed that the internal stcE probe hybridized to
both plasmid DNA and contaminating linear DNA fragments
(consisting of both sheared plasmid and chromosomal DNA)
present in the preparations of EDL933 and DEC 5A, 5C, and
5E (figure 2). This reveals that stcE is located on a plasmid in
DEC 5A, 5C, and 5E. Although a second copy of stcE is absent
from the genome sequences of EDL933 [7] and the E. coli O157:
H7 strain from the Sakai outbreak [35], we cannot exclude the
possibility that an additional copy of the gene exists on the
chromosome of the DEC 5 strains because of the hybridization
of the linear DNA fragments. As expected, no signal was evident
in preparations from EDL933cu (lacking pO157), DEC 5B, and
DEC 5D.
Prevalence of other pO157 markers among diarrheagenic
E. coli. In addition to stcE, genes for other potential virulence
factors are located on pO157 [14]. These include the ehx operon
encoding the EHEC hemolysin [16]; espP, an extracellular serine
protease [18]; and katP, a periplasmic catalase-peroxidase [19].
pO157 also contains 13 genes that encode a type II secretion
cluster, etpC–etpO [22]. The etp cluster secretes StcE, and disruption of the gene encoding the putative outer-membrane
component of the apparatus, etpD, prevents the release of StcE
from the bacterium [23]. We screened the DEC collection and
other DEC strains for the presence of ehxA, espP, katP, and
etpD by PCR or colony blot to examine the distribution of the
genes and to determine whether any are linked to stcE. As
expected, EDL933 and all O157:H7 strains in the DEC collection carried all 5 genes. However, CDC G5101, an O157:H7
strain with a unique stcE restriction fragment–length polymorphism pattern, contained etpD and ehxA but lacked espP
and katP (table 4). The O157:H⫺ strains tested also carried stcE,
etpD, and ehxA but lacked espP and katP, whereas the O55:H7
Figure 1. Production and activity of StcE from Escherichia coli strains EDL933; EDL933cu; and DEC 3A–3E, DEC 4A–4E, and DEC 5A–5E from the
diarrheagenic E. coli (DEC) collection. A, Immunoblot analysis of concentrated culture supernatants (30 mL), using a polyclonal anti-StcE antibody; the
lower band is nonspecific. B, Immunoblot analysis of full-length C1 esterase inhibitor (C1-INH) after incubation with concentrated culture supernatants,
using a polyclonal anti–C1-INH antibody.
strains either lacked all 5 markers (TB156A and DEC 5B and
5D) or contained only stcE and etpD (5905, TB182A, and DEC
5A, 5C, and 5E). None of the O55:H7 strains we examined
encoded ehxA, espP, or katP. Similarly, ECOR37, a related an-
cestor of E. coli O55:H7, carried stcE and etpD but not ehxA,
espP, or katP. The other serotypes in the DEC collection contained a heterogeneous collection of ehxA, espP, and katP, but
all lacked etpD (table 4). In fact, etpD was only present in strains
Figure 2. Detection of plasmidborne stcE DNA in strains EDL933, EDL933cu, and DEC 5A–5E from the diarrheagenic Escherichia coli (DEC) collection.
Plasmid DNA was isolated from strains and electrophoresed on a 1% agarose gel (left panel). DNA was transferred to nylon and probed with a 927bp 32P-labeled DNA fragment of stcE (right panel).
Acquisition of stcE by E. coli O157:H7 • JID 2003:187 (15 June) • 1911
Table 4.
Prevalence of multiple pO157-associated markers
among Escherichia coli strains.
Strain
Serotype
stcE
etpD
ehxA
espP
katP
DEC 1A–1E
DEC 2A–2E
O55:H6
⫺
⫺
⫺
⫺
⫺
O55:H6
⫺
⫺
⫺
⫺
⫺
+
EDL933
O157:H7
+
+
+
+
DEC 3A–3E
O157:H7
+
+
⫺
+
+
DEC 4A–4E
O157:H7
+
+
⫺
+
+
CDC G5101
O157:H7
+
+
+
⫺
⫺
⫺
493/89
O157:H
+
+
+
⫺
FDA 413
O157:NM
⫺
⫺
⫺
⫺
⫺
2937
O157:H⫺
+
+
+
⫺
⫺
6790
O157:H⫺
+
+
+
⫺
⫺
431
O157:H
⫺
+
+
+
⫺
⫺
659
O157:H⫺
+
+
+
⫺
⫺
2576
O157:H⫺
+
+
+
⫺
⫺
5905
O55:H7
+
+
⫺
⫺
⫺
DEC 5A
O55:H7
+
+
⫺
⫺
⫺
DEC 5B
O55:H7
⫺
⫺
⫺
⫺
⫺
DEC 5C
O55:H7
+
+
⫺
⫺
⫺
DEC 5D
O55:H7
⫺
⫺
⫺
⫺
⫺
DEC 5E
O55:H7
+
+
⫺
⫺
⫺
TB182A
O55:H7
+
+
⫺
⫺
⫺
TB156A
O55:H7
⫺
⫺
⫺
⫺
⫺
ECOR37
ON:H7
+
+
⫺
⫺
⫺
DEC 6A–6E
O111:H12
⫺
⫺
⫺
1/5a
⫺
DEC 7A–7E
O157:H43
⫺
⫺
⫺
DEC 8A–8E
O111:H8
⫺
⫺
4/5
DEC 9A–9E
O26:H11
⫺
⫺
⫺
⫺
⫺
DEC 10A–10E
O26:H11
⫺
⫺
4/5a
4/5a
4/5a
DEC 11A–11E
O128:H2
⫺
⫺
1/5
DEC 12A–12E
O111:H2
⫺
⫺
⫺
⫺
⫺
DEC 13A–13E
O128:H7
⫺
⫺
⫺
⫺
⫺
DEC 14A–14E
O128:H21
⫺
⫺
⫺
⫺
⫺
DEC 15A–15E
O111:H21
⫺
⫺
⫺
⫺
⫺
⫺
⫺
a
a
4/5
1/5
⫺
a
a
4/5
a
⫺
NOTE.
Strains were examined for the presence of genes found on
pO157, including stcE, etpD, ehxA, espP, and katP, by colony blot or polymerase chain reaction, as described in Materials and Methods. Strains of
the serotype O157:H7 universally carry all 5 markers, whereas other serotypes contain a heterogeneous collection of these genes. Strains that carry
stcE also carry etpD, and strains that lack stcE also lack etpD. DEC, diarrheagenic E. coli; +, positive/present; ⫺, negative/not present.
a
No. of strains carrying gene/total no. tested.
that carried stcE, and vice versa. This suggests that the genes
for stcE and the etp operon are linked.
DISCUSSION
Based on our survey of the DEC collection, StcE, a metalloprotease secreted by EHEC EDL933, is limited to a subset of
DEC strains. Specifically, colony blot analysis of 74 isolates in
1912 • JID 2003:187 (15 June) • Lathem et al.
the DEC collection revealed that only strains of the serotypes
O157:H7 and O55:H7 contained sequences that hybridized with
a 927-bp region of stcE. Although all O157:H7 strains were
positive, only 3 of 5 O55:H7 strains carried the gene (table 3).
An examination of a large set of EHEC clinical isolates from
the upper Midwest established that stcE is common to most
O157:H7 strains tested. This is not surprising, because E. coli
O157:H7 is thought to be clonal in nature [36]. However, the
absence of stcE among other STEC serotypes not closely allied
with the O157:H7 clonal group suggests that stcE is limited to
the EHEC 1 complex.
The stcE-positive strains in the DEC collection were tested
for their ability to release an active protease into the culture
supernatant. The supernatants of all O157:H7 strains and 2 of
3 O55:H7 strains were able to degrade C1-INH and contained
an appropriately sized polypeptide that reacted with a polyclonal anti-StcE antibody (figure 1). This demonstrates that
these strains produce StcE and supports the clonal nature of
the group. It also suggests that these strains contain a secretion
system for StcE. Based on the ability of the etp type II secretion
pathway to secrete StcE from EDL933, it seems likely that the
etp genes, if present, are responsible for StcE secretion in these
strains. In fact, all stcE-positive strains that we examined also
contained the gene for etpD, the putative outer-membrane
component of the etp type II secretion system. No strain we
tested carried one gene and not the other; it appears that stcE
and the etp cluster are linked and were acquired by E. coli at
the same point. The one O55:H7 strain (DEC 5C) that contained stcE-like DNA but did not release a StcE-reactive polypeptide into the culture supernatant or degrade C1-INH may
have a defect in the transcription or translation of stcE. Alternatively, DEC 5C may produce but not release StcE because of
a nonfunctional secretion mechanism.
By using multilocus enzyme electrophoresis to determine ET,
Feng et al. [9] postulated a model of stepwise evolution of E.
coli O157:H7, beginning with a common O55:H7 ancestor. The
model predicts the acquisition of virulence factors and loss or
transition of function, including a serotype shift from O55 to
O157, at defined points that result in the emergence of the virulent O157:H7 serotype. The presence of stcE in some O55:H7
strains and ECOR37 led us to examine the presence of other
plasmidborne virulence factors associated with E. coli O157:H7
to determine the points at which they were acquired during the
evolution of O157:H7. We examined 4 additional genes with
established or hypothesized functions: etpD, ehxA, espP, and katP.
We determined that the acquisition of plasmidborne EHEC virulence factors began with the gain of stcE and etpD by ECOR37,
a closely related member of the EHEC 1 complex, or an earlier
ancestor. The initial O55:H7 group (ET 5, ancestral cell A1)
subsequently can be divided into 2 subgroups. The first is represented by strains DEC 5A, 5C, and 5E and TB182A, in which
Figure 3. Evolutionary model of the emergence of Escherichia coli O157:H7, based on the acquisition of described virulence factors. Branch lengths
of the cladogram are arbitrary and are not set to an evolutionary scale. Representative strains are listed to the right of each terminal branch. Arrows
indicate gains and losses of genes or phenotypes. Dashed lines indicate hypothesized changes. DEC, diarrheagenic E. coli; ?, not isolated.
stcE and etpD are present, followed by the second O55:H7 subgroup, in which the 2 genes were lost, represented by strains
DEC 5B and 5D and TB156A (table 4). Alternatively, is possible
that ECOR37 and E. coli O55:H7 acquired stcE and etpD independently. However, the first premise is more likely, judging by
the phylogenetic similarity between ECOR37 and E. coli O55:H7
[37, 38], their identical stcE restriction fragment–length polymorphism patterns (data not shown), and the absence of other
plasmidborne virulence markers (table 4). This also suggests that
a portion of the EHEC plasmid was acquired earlier than originally predicted [4], because stcE is located on a plasmid in the
DEC 5 strains (figure 2).
The acquisition of ehxA presumably occurred during the
transition from O55 to O157 antigen. At this point, the lineage
diverges, into one line in which motility was lost and another
in which additional virulence factors were acquired. The nonmotile branch, represented by strains 493/89 and 2937, retained
stcE, etpD, and ehxA but did not acquire espP and katP. Previous work from Schmidt et al. [31] showed that stx-negative
O157:H⫺ strains are ehx and etp positive but espP and katP
negative, which supports the hypothesized evolution of this
branch. The primary branch, however, acquired stx1 (strain
CDC G5101), followed by espP and katP (DEC 3 and 4 and
EDL933). Although grouped with the A6 strains [9], FDA 413
lacks all 5 plasmidborne virulence traits. This is likely the result
of the recent loss of the plasmid in nature or during isolation
and culture. We hypothesize that stcE and etp were acquired
early in the evolution of O157:H7, before the acquisition of
other plasmidborne virulence factors, such as espP, katP, and
ehxA, and before the inheritance of the stx prophage (figure
3). Our data also underscore the diversity among EPEC strains
and, more notably, that some EPEC isolates carry a putative
EHEC virulence factor.
Although the role of stcE in the pathogenesis of EHEC remains unclear, we have demonstrated that stcE is carried by E.
coli strains other than the current O157:H7 serotype. However, a search of the GenBank database [39] shows no homologues
to StcE, with the exception of TagA from Vibrio cholerae, a
ToxR-regulated protein of unknown function [40]. A 301-aa
segment of StcE shares 42% identity with 304 aa of TagA that
span the metalloprotease active site. Whether stcE was horizontally acquired from V. cholerae, whether tagA transferred
from E. coli, or whether both genes came from a third, unidentified source is unknown. An assessment of potential TagA
metalloprotease activity is ongoing. In sum, our data describe
the evolving nature of the EHEC plasmid, beginning early in
the evolution of E. coli O157:H7, and support the stepwise
evolution hypothesis put forth by Feng et al. [9].
References
1. Donnenberg MS, Whittam TS. Pathogenesis and evolution of virulence
in enteropathogenic and enterohemorrhagic Escherichia coli. J Clin Invest 2001; 107:539–48.
2. Lior H. Escherichia coli O157:H7 and verotoxigenic Escherichia coli
(VTEC). Dairy Food Environ Sanit 1994; 14:378–82.
3. Whittam TS. Evolution of Escherichia coli O157:H7 and other Shiga
toxin–producing E. coli strains. In: Kaper JB, O’Brien AD, eds. Escherichia coli O157:H7 and other Shiga toxin–producing E. coli strains.
Washington, DC: American Society for Microbiology, 1998:195–209.
Acquisition of stcE by E. coli O157:H7 • JID 2003:187 (15 June) • 1913
4. Levine MM, Xu JG, Kaper JB, et al. A DNA probe to identify enterohemorrhagic Escherichia coli of O157:H7 and other serotypes that cause
hemorrhagic colitis and hemolytic uremic syndrome. J Infect Dis 1987;
156:175–82.
5. Whittam TS, Wolfe ML, Wachsmuth IK, Orskov F, Orskov I, Wilson
RA. Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea. Infect Immun 1993; 61:1619–29.
6. Reid SD, Herbelin CJ, Bumbaugh AC, Selander RK, Whittam TS. Parallel evolution of virulence in pathogenic Escherichia coli. Nature 2000;
406:64–7.
7. Perna NT, Plunkett G 3rd, Burland V, et al. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 2001; 409:529–33.
8. Rodrigues J, Scaletsky IC, Campos LC, Gomes TA, Whittam TS, Trabulsi LR. Clonal structure and virulence factors in strains of Escherichia
coli of the classic serogroup O55. Infect Immun 1996; 64:2680–6.
9. Feng P, Lampel KA, Karch H, Whittam TS. Genotypic and phenotypic changes in the emergence of Escherichia coli O157:H7. J Infect Dis
1998; 177:1750–3.
10. Ostroff SM, Tarr PI, Neill MA, Lewis JH, Hargrett-Bean N, Kobayashi
JM. Toxin genotypes and plasmid profiles as determinants of systemic
sequelae in Escherichia coli O157:H7 infections. J Infect Dis 1989; 160:
994–8.
11. Fratamico PM, Bhaduri S, Buchanan RL. Studies on Escherichia coli serotype O157:H7 strains containing a 60-MDa plasmid and on 60-MDa
plasmid–cured derivatives. J Med Microbiol 1993; 39:371–81.
12. Paros M, Tarr PI, Kim H, Besser TE, Hancock DD. A comparison of
human and bovine Escherichia coli O157:H7 isolates by toxin genotype,
plasmid profile, and bacteriophage lambda–restriction fragment length
polymorphism profile. J Infect Dis 1993; 168:1300–3.
13. Brunder W, Schmidt H, Frosch M, Karch H. The large plasmids of Shigatoxin–producing Escherichia coli (STEC) are highly variable genetic elements. Microbiology 1999; 145:1005–14.
14. Burland V, Shao Y, Perna NT, Plunkett G, Sofia HJ, Blattner FR. The
complete DNA sequence and analysis of the large virulence plasmid
of Escherichia coli O157:H7. Nucleic Acids Res 1998; 26:4196–204.
15. Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev
1998; 11:142–201.
16. Bauer ME, Welch RA. Characterization of an RTX toxin from enterohemorrhagic Escherichia coli O157:H7. Infect Immun 1996; 64:167–75.
17. Schmidt H, Beutin L, Karch H. Molecular analysis of the plasmidencoded hemolysin of Escherichia coli O157:H7 strain EDL 933. Infect
Immun 1995; 63:1055–61.
18. Brunder W, Schmidt H, Karch H. EspP, a novel extracellular serine
protease of enterohaemorrhagic Escherichia coli O157:H7 cleaves human coagulation factor V. Mol Microbiol 1997; 24:767–78.
19. Brunder W, Schmidt H, Karch H. KatP, a novel catalase-peroxidase
encoded by the large plasmid of enterohaemorrhagic Escherichia coli
O157:H7. Microbiology 1996; 142:3305–15.
20. Klapproth JM, Donnenberg MS, Abraham JM, James SP. Products of
enteropathogenic E. coli inhibit lymphokine production by gastrointestinal lymphocytes. Am J Physiol 1996; 271:G841–8.
21. Tatsuno I, Horie M, Abe H, et al. toxB gene on pO157 of enterohemorrhagic Escherichia coli O157:H7 is required for full epithelial cell
adherence phenotype. Infect Immun 2001; 69:6660–9.
22. Schmidt H, Henkel B, Karch H. A gene cluster closely related to type
II secretion pathway operons of gram-negative bacteria is located on
the large plasmid of enterohemorrhagic Escherichia coli O157 strains.
FEMS Microbiol Lett 1997; 148:265–72.
23. Lathem WW, Grys TE, Witowski SE, et al. StcE, a metalloprotease se-
1914 • JID 2003:187 (15 June) • Lathem et al.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
creted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor. Mol Microbiol 2002; 45:277–88.
Caliezi C, Wuillemin WA, Zeerleder S, Redondo M, Eisele B, Hack CE.
C1-esterase inhibitor: an anti-inflammatory agent and its potential use
in the treatment of diseases other than hereditary angioedema. Pharmacol Rev 2000; 52:91–112.
Elliott SJ, Sperandio V, Giron JA, et al. The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and
non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun 2000; 68:6115–26.
Karch H, Heesemann J, Laufs R, O’Brien AD, Tacket CO, Levine MM.
A plasmid enterohemorrhagic Escherichia coli O157:H7 is required for
expression of a new fimbrial antigen and for adhesion to epithelial cells.
Infect Immun 1987; 55:455–61.
Ochman H, Selander RK. Standard reference strains of Escherichia coli
from natural populations. J Bacteriol 1984; 157:690–3.
Hayes PS, Blom K, Feng P, Lewis J, Strockbine NA, Swaminathan B.
Isolation and characterization of a beta-d-glucuronidase–producingstrain
of Escherichia coli serotype O157:H7 in the United States. J Clin Microbiol
1995; 33:3347–8.
Karch H, Bohm H, Schmidt H, Gunzer F, Aleksic S, Heesemann J.
Clonal structure and pathogenicity of Shiga-like toxin–producing, sorbitol-fermenting Escherichia coli O157:H. J Clin Microbiol 1993; 31:
1200–5.
Wieler LH, Tigges M, Ebel F, et al. The enterohemolysin phenotype
of bovine Shiga-like toxin–producing Escherichia coli (SLTEC) is encoded by the EHEC-hemolysin gene. Vet Microbiol 1996; 52:153–64.
Schmidt H, Scheef J, Huppertz HI, Frosch M, Karch H. Escherichia coli
O157:H7 and O157:H⫺ strains that do not produce Shiga toxin: phenotypic and genetic characterization of isolates associated with diarrhea
and hemolytic-uremic syndrome. J Clin Microbiol 1999; 37:3491–6.
Schmidt H, Geitz C, Tarr PI, Frosch M, Karch H. Non-O157:H7 pathogenic Shiga toxin–producing Escherichia coli: phenotypic and genetic profiling of virulence traits and evidence for clonality. J Infect Dis
1999; 179:115–23.
Malstrom C, James S. Inhibition of murine splenic and mucosal lymphocyte function by enteric bacterial products. Infect Immun 1998; 66:
3120–7.
Welch RA, Hull R, Falkow S. Molecular cloning and physical characterization of a chromosomal hemolysin from Escherichia coli. Infect
Immun 1983; 42:178–86.
Hayashi T, Makino K, Ohnishi M, et al. Complete genome sequence
of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. DNA Res 2001; 8:11–22.
Whittam TS, Wachsmuth IK, Wilson RA. Genetic evidence of clonal
descent of Escherichia coli O157:H7 associated with hemorrhagic colitis
and hemolytic uremic syndrome. J Infect Dis 1988; 157:1124–33.
Brown EW, LeClerc JE, Li B, Payne WL, Cebula TA. Phylogenetic evidence for horizontal transfer of mutS alleles among naturally occurring
Escherichia coli strains. J Bacteriol 2001; 183:1631–44.
Pupo GM, Karaolis DK, Lan R, Reeves PR. Evolutionary relationships
among pathogenic and nonpathogenic Escherichia coli strains inferred
from multilocus enzyme electrophoresis and mdh sequence studies. Infect
Immun 1997; 65:2685–92.
Altschul SF, Madden TL, Schaffer AA, et al. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic
Acids Res 1997; 25:3389–402.
Peterson KM, Mekalanos JJ. Characterization of the Vibrio cholerae ToxR
regulon: identification of novel genes involved in intestinal colonization.
Infect Immun 1988; 56:2822–9.