EspP, aType V-secreted serine protease of enterohaemorrhagic
Escherichia coli O157:H7, in£uences intestinal colonization of calves
and adherence to bovine primary intestinal epithelial cells
Francis Dziva1, Arvind Mahajan2, Pamela Cameron2, Carol Currie2, Iain J. McKendrick3,
Timothy S. Wallis1, David G.E. Smith2,4 & Mark P. Stevens1
1
Division of Microbiology, Institute for Animal Health, Compton, Newbury, Berkshire, UK; 2Functional Genomics Unit, Moredun Research Institute,
Pentland Science Park, Penicuik, Midlothian, UK; 3Biomathematics & Statistics Scotland (BioSS), James Clerk-Maxwell Building, Edinburgh, UK; and
4
Institute for Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Glasgow, UK
Correspondence: Francis Dziva, Division of
Microbiology, Institute for Animal Health,
Compton, Newbury, Berkshire, RG20 7NN,
UK. Tel.: 144 1635 578 411; fax: 144 1635
577 237; e-mail: francis.dziva@bbsrc.ac.uk
Present address: Timothy S. Wallis,
Ridgeway Biologicals Ltd, c/o Institute for
Animal Health, Compton, Newbury, Berkshire
RG20 7NN, UK.
Received 12 February 2007; accepted 14 March
2007.
Abstract
Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of zoonotic
diarrhoeal pathogens of worldwide importance. Cattle are a key reservoir; however
the molecular mechanisms that promote persistent colonization of the bovine
intestines by EHEC are ill-defined. The large plasmid of EHEC O157:H7 encodes
several putative virulence factors. Here, it is reported that the pO157-encoded Type
V-secreted serine protease EspP influences the intestinal colonization of calves. To
dissect the basis of attenuation, a bovine primary rectal epithelial cell line was
developed. Adherence of E. coli O157:H7 to such cells was significantly impaired by
espP mutation but restored upon addition of highly purified exogenous EspP. Data
of this study add to the growing body of evidence that cytotoxins facilitate
intestinal colonization by EHEC.
DOI:10.1111/j.1574-6968.2007.00724.x
Editor: Rob Delahay
Keywords
Escherichia coli ; EspP; cattle; adherence;
colonization.
Introduction
Enterohaemorrhagic Escherichia coli (EHEC) pose a significant public health threat worldwide. Ruminants are a key
reservoir and humans are frequently infected following
direct or indirect exposure to ruminant faeces. Strategies to
reduce the carriage of EHEC in ruminants are expected to
lower the incidence of human disease. However, crossprotective vaccines do not yet exist and the molecular
mechanisms that influence the persistence of EHEC in the
bovine intestines are incompletely understood. By allelic
exchange and signature-tagged mutagenesis (STM), portfolios of bacterial genes that influence colonization of calves by
EHEC O157:H7 and O26:H-have been identified, including
conserved and serotype-specific factors (Dziva et al., 2004;
van Diemen et al., 2005). Among the EHEC O26:H- genes
putatively required for the intestinal colonization of calves is
pssA (van Diemen et al., 2005). PssA (protein secreted by
FEMS Microbiol Lett xx (2007) 000–000
Shiga toxin-producing E. coli) was first described in EHEC
O26 strains and is highly related to a family of serine
protease autotransporters of Enterobacteriaceae (SPATEs;
Djafari et al., 1997). Parallel studies on EHEC O157:H7
identified a pO157-encoded secreted serine protease (EspP)
which cleaves human coagulation factor V (Brunder et al.,
1997). Through hybridization and immunoblot assays, EspP
was shown to be widespread in EHEC O157:H7 strains and
to exist in EHEC O26 strains (Brunder et al., 1997) and it is
now clear that PssA and EspP are 99% identical at the amino
acid level. Despite this close homology, PssA has been
reported to cleave casein and exhibit cytopathic activity for
Vero cells, phenotypes that have not been reported with
EspP (Djafari et al., 1997; Dutta et al., 2002).
Several SPATEs have been extensively characterized
in vitro, including EspC of enteropathogenic E. coli (EPEC;
Stein et al., 1996; Navarro-Garcia et al., 2004), Pet of
enteroaggregative E. coli (EAEC; Eslava et al., 1998), EatA
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c
2
of enterotoxigenic E. coli (ETEC; Patel et al., 2004), SigA of
Shigella flexneri (Al-Hasani et al., 2000), and the uropathogenic E. coli (UPEC) proteins Sat (Guyer et al., 2000) and
PicU (Parham et al., 2004). In rabbit ileal loop models,
mutation of sat (of diffusely adhering E. coli), ETEC eatA
and Shigella SigA impaired the induction of intestinal
inflammatory and secretory responses (Al-Hasani et al.,
2000; Patel et al., 2004; Taddei et al., 2005). Furthermore,
EAEC Pet causes damage to human intestinal explants
cultured ex vivo (Henderson et al., 1999) and EPEC EspC is
toxic to rat jenunal explants (Mellies et al., 2001). Taken
together, these observations indicate that SPATEs may play
key roles in host–pathogen interactions at epithelial surfaces
(reviewed in Henderson et al., 2004).
The majority of SPATEs are encoded on large plasmids. In
addition to EspP, the large plasmid of EHEC O157:H7
(pO157) encodes enterohaemolysin (EhxA), catalase-peroxidase (KatP), a large clostridial toxin homologue (ToxB), a
metalloprotease that cleaves C1 esterase inhibitor (StcE,
initially identified as TagA) and the ecf operon (Burland
et al., 1998; Makino et al., 1998). It has been reported that
pO157 is required for full adherence to epithelial cells (Toth
et al., 1990) and ToxB and StcE have since been implicated in
this process (Tastuno et al., 2001; Stevens et al., 2004; Grys
et al., 2005). Although it was initially reported that pO157
does not influence E. coli O157:H7-induced enteritis and
adherence in gnotobiotic piglets (Tzipori et al., 1987), it was
recently reported that pO157 influences colonization of the
bovine terminal rectum (Sheng et al., 2006) and that the ecf
operon is required for persistence in the bovine intestines
(Yoon et al., 2005). With the exception of toxB, which is not
required for colonization of young calves (Stevens et al.,
2004), the role of other pO157-encoded genes in vivo has
not been defined. Evidence for expression during E. coli
O157:H7 infection of humans has been obtained for EspP
(Brunder et al., 1997), EhxA (Schmidt et al., 1995), StcE
(Paton & Paton, 2002) and a pO157-encoded inner membrane acyltransferase (MsbB2; John et al., 2005).
Here, it is shown that EspP contributes to adherence to
bovine primary rectal cells and colonization of the bovine
intestines by the predominant EHEC serotype affecting
humans in Europe and North America.
F. Dziva et al.
expression and purification of EspP. Bacterial strains were
grown in Luria–Bertani (LB) broth or agar supplemented
with ampicillin (Amp, 100 mg mL 1), kanamycin (Kan,
50 mg mL 1) and nalidixic acid (Nal, 25 mg mL 1) as appropriate. For adherence assays, bacteria were grown in minimum essential medium buffered with HEPES (MEMHEPES; Sigma, St Louis).
Construction and characterization of an E. coli
O157:H7 DespP::kanR mutant
The espP gene was replaced by a kanamycin resistance
cassette by integration of a linear PCR product upon
transient expression of lRed recombinase, essentially as
described (Datsenko & Wanner, 2000). The amplicon was
generated using VentTM proofreading DNA polymerase
(New England Biolabs, Hitchin, UK) and primers espP1FFRT (5 0 -GATAAACATACTGTGTTTGTTATCCGTATGATA
ACAAACACATAAAGGAGGTGTAGGCTGGAGCTGCTTC
-3 0 ), and espP2-FRT (5 0 -CGGCAGGCACTGAGGGTAAAG
GGCCCGCAGGCCCTTTTGAATACGGAGTACATATGAA
TATCCTCCTTAG-3 0 ) which contain 50 nucleotide homology extensions corresponding to the regions immediately 5 0
and 3 0 of espP and priming sequences for the kanR cassette of
pKD4 (bold; Datsenko & Wanner, 2000). The PCR product
was DpnI digested to remove residual template, gel purified
and electroporated into 85-170nalR carrying the temperature-sensitive lRed helper plasmid pKD46 following induction of lRed recombinase with 10 mM L-arabinose at 30 1C.
A recombinant (85-170nalR DespP::kanR) was selected on LB
agar containing nalidixic acid and kanamycin and cured of
pKD46 by growth at 42 1C. The insertion was confirmed by
PCR using the espP flanking primers espP-F (5 0 -CTCCTTTA
CTGTATCCCTCAT-3 0 ) and espP-R (5 0 -AGGCACTGAGGG
TAAAGG-3 0 ).
To verify the mutant, 85-170nalR and 85-170nalR DespP::kanR were cultivated in Dulbecco’s Modified Eagles Medium
(DMEM) to induce protein secretion and secreted proteins
were prepared by trichloroacetic acid precipitation (Stevens
et al., 2004). Secreted proteins were analysed by 4–15%
gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized following staining
with GelCode Blue reagent (Perbio Science UK Ltd,
Cheshire, UK).
Materials and methods
Bovine primary cell culture
Bacterial strains and media
Strain 85-170nalR is a nalidixic acid-resistant nontoxigenic
E. coli O157:H7 strain that reliably colonizes the intestines of
calves and induces AE lesions in vivo (Stevens et al., 2004;
Vlisidou et al., 2006a). The laboratory E. coli strain HB101
was transformed with plasmid pB9-5espP (kindly supplied
by Anthony Scott-Tucker, University of Birmingham) for
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Published by Blackwell Publishing Ltd. All rights reserved
Bovine primary epithelial cells were derived from the region 0
to 5 cm proximal to the rectal-anal junction of cattle up to 30
months old using a modification of the method described for
colonic epithelial cultures (Hoey et al., 2003). Unless stated
otherwise, reagents for the procedure were obtained from
Sigma-Aldrich. Briefly, mucosal scrapings from the terminal
rectum were digested in DMEM containing 1% (v/v) foetal
FEMS Microbiol Lett xx (2007) 000–000
3
EspP influences intestinal colonization and adherence by E. coli
calf serum (FCS), 100 U mL 1 penicillin, 30 mg mL 1 streptomycin, 25 mg mL 1 gentamicin, 75 U mL 1 collagenase and
20 mg mL 1 dispase (Roche, Rockford) with gentle shaking at
37 1C until isolated crypts could be observed microscopically.
A series of differential centrifugation steps with DMEM
containing 2% (w/v) sorbitol was used to enrich the isolated
crypts from undigested material, endogenous microbial communities and single cells including fibroblasts (Booth et al.,
1995). The crypt cell pellet was resuspended in cell culture
medium [DMEM, 2.5% (v/v) FCS, 0.25 U mL 1 insulin,
10 ng mL 1 epidermal growth factor (EGF) and 30 mg mL 1
gentamicin]. Approximately 400–600 crypts were seeded per
well into 24-well Costar culture plates (Corning, Cambridge)
precoated with collagen (Vitrogen Collagen, Nutacon, Netherlands). To inhibit fibroblast growth, the medium was
replaced with MEM D-Valine medium containing 10% (v/v)
batch-tested FCS, 0.25 U mL 1 insulin, 10 ng mL 1 EGF and
30 mg mL 1 gentamicin after 24 h as described previously
(Hoey et al., 2003). The cells were grown to confluence
(c. 3 105 cells per well, typically achieved 10–14 days after
isolation). The cultured cells were confirmed by immunocytochemistry to possess epithelial cell-specific cytokeratins
(CK4, 5, 6, 8, 10, 13 and 18) and lack a marker for fibroblasts
as described (Hoey et al., 2003).
Purification of exogenous EspP
Purified recombinant EspP was prepared essentially as
described (Navarro-Garcia et al., 2004). Briefly, the EspPoverproducing strain HB101(pB9-5 espP) was grown in LB
overnight at 37 1C and the supernatant harvested following
centrifugation. The culture supernatant was filtered through
a 0.22 mm pore-size low-protein-binding membrane, then
concentrated by centrifugation in against a Centricon filter
with a 100 kDa cut-off (Millipore, Bedford). The retentate
was reconstituted in MEM-HEPES, checked for purity by
SDS-PAGE and confirmed to contain EspP by Western
blotting. The amount of protein was estimated using the
standard BCA method (Pierce Biotechnology Inc, Rockford)
and stored at 20 1C until required.
washing three times with phosphate-buffered saline (PBS)
and adherent viable bacteria were enumerated following
dispersion with PBS-0.1% (v/v) Triton X-100, serial 10-fold
dilution and plating onto selective media. The assays were
carried out independently on three occasions and the data
analysed with a generalized linear mixed model (GLMM)
assuming a Poisson response variable (McCullagh & Nelder,
1989). The canonical link function was used. The experimental replicate was fitted as a random effect. Strain and the
presence/absence of exogenous EspP were fitted as fixed
effects. Evaluation of the significance of fixed effects was
carried out using the Chi-squared approximation for the
Wald test.
Oral infection of calves with E. coli O157:H7
Procedures for oral inoculation of calves with E. coli
O157:H7 have been described (Stevens et al., 2004) and
comply with the Animals (Scientific Procedures) Act 1986
(licence 30/2009). Three 12-day-old Friesian bull calves were
coinfected with c. 1 1010 CFU each of 85-170nalR and 85170nalR DespP::kanR grown to stationary phase in brain
heart infusion broth in a 1 : 1 ratio. The magnitude and
duration of faecal excretion of the bacteria were followed at
least once daily for 12 days by plating of triplicate serial
dilutions of fresh faeces collected by rectal palpation to
sorbitol MacConkey agar (Oxoid, Basingstoke, UK) supplemented with 2.5 mg mL 1 potassium tellurite and 20 mg mL 1
nalidixic acid (T–SMAC–Nal) and T–SMAC–Nal containing
50 mg mL 1 kanamycin (T–SMAC–Nal–Kan). The number
of wild-type bacteria was calculated by subtracting the viable
count on T–SMAC–Nal–Kan from that obtained using
T–SMAC–Nal as previously described (Dziva et al., 2004).
Recoveries of wild-type and the mutant bacteria were
confirmed by PCR from selected colonies using espP-flanking primers. The faecal shedding data were analysed for the
effect of mutation by means of an F-test, with the data taken
as repeated measurements and the animal as a covariant
(Proc Mixed, Statistical Analysis System 1995, SAS Institute,
Cary). P values o 0.05 were taken to be significant.
Adherence assays
Stationary-phase LB cultures of wild-type and the mutant
bacteria grown in MEM-HEPES with appropriate antibiotics were diluted 1 : 10 in MEM-HEPES and incubated at
37 1C with shaking (200 r.p.m.) to an OD at 600 nm of
0.3–0.4. Confluent bovine rectal primary epithelial cells
(above) were separately infected in duplicate with each of
the strains at a multiplicity of infection of c. 100 in MEMHEPES and incubated for 1 h at 37 1C in a humidified 5%
CO2 atmosphere. Purified recombinant EspP was supplemented to a final concentration of 178.8 ng mL 1 of protein
where appropriate. Nonadherent bacteria were removed by
FEMS Microbiol Lett xx (2007) 000–000
Results and discussion
Characterization of an E. coli O157:H7 espP::kanR
mutant
A espP::kanR mutant of E. coli O157:H7 strain 85-170nalR
was constructed by lRed-mediated linear recombination
and verified by PCR with espP-flanking primers (data not
shown) and SDS-PAGE analysis of proteins secreted by the
wild-type and mutant strains. While both the mutant and
parent strains secreted comparable quantities of the Type III
secreted proteins EspA, -B, -D and Tir, a protein of the
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Published by Blackwell Publishing Ltd. All rights reserved
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4
F. Dziva et al.
expected size of EspP was absent in the secreted fraction of
the mutant (Fig. 1). The protein present in the secreted
proteome of the wild-type at this position has been independently confirmed to be EspP by tandem MS (Roe et al.,
2007). The growth rate of the 85-170nalR espP::kanR mutant
was confirmed to be identical to that of the parent strain in
both rich and minimal media by real-time spectrophotometry (data not shown).
1
2
116
EspP
97
66
55
EspD
EspB
36
31
EspA
21
Fig. 1. Characterization of a DespP::kanR mutant of Escherichia coli
O157:H7 strain 85-170nalR. Precipitated secreted proteins from the
parent (lane 1) and DespP::kanR mutant (lane 2) strains were analysed
by SDS-PAGE.
EspP plays a role in the intestinal colonization of
calves by E. coli O157:H7
The role of EspP in the intestinal colonization of calves was
determined using coinfection experiments, using a model
that has reliably identified attenuating defects previously
(Stevens et al., 2002; Dziva et al., 2004; Vlisidou et al.,
2006a, b). From day 5 postoral inoculation, the 85-170nalR
DespP::kanR mutant was shed in the faeces in lower numbers
than the parent strain and the reduction became statistically
significant from day 9 onwards (P values o 0.05; Fig. 2).
Although the role of secreted serine protease in persistence
of the bovine intestines was first suggested by the isolation of
two independent signature-tagged mini-Tn5Km2 mutants
of EHEC O26:H- with insertions in pssA (van Diemen et al.,
2005), it has been observed that some genes identified by
STM are not required for persistence when defined mutants
are constructed and screened in isolation or by coinfection
in the same model (map; Dziva et al., 2004; nleD; Marchés
et al., 2005). Thus, the data herein provide novel independent validation of the role of EspP in intestinal colonization.
The extent of attenuation caused by the espP mutation was
slight compared with the effect of intimin and Tir mutations
in the same strain in calves of the same age (Vlisidou et al.,
2006a), but is significant when compared with an 85170nalR mutant lacking the putative cytotoxin/adhesin ToxB
(Stevens et al., 2004).
Although it has been shown that EspP is required for
intestinal colonization of cattle by E. coli O157:H7, it is
noteworthy that sorbitol-fermenting EHEC O157:H-strains
isolated from cattle lack espP (Brunder et al., 1999; Karch &
9
8
Log10 count g–1 faeces
7
6
5
4
3
2
1
0
0
1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8
Days postinfection
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Published by Blackwell Publishing Ltd. All rights reserved
9 10 11 12
Fig. 2. Course of faecal excretion of Escherichia
coli O157:H7 strain 85-170nalR following
coinfection of calves (n = 3) with the parent
strain (-B-) and an isogenic DespP::kanR
mutant (-’-). Data represent the mean daily
faecal count SE of the mean.
FEMS Microbiol Lett xx (2007) 000–000
5
EspP influences intestinal colonization and adherence by E. coli
Bielaszewska, 2001) and a mutated form of espP was reported
in five of 30 bovine E. coli O157:H7 isolates (McNally et al.,
2001). However, the possibility of redundancy in the repertoire of secreted proteases in these strains cannot be precluded. Data of this study add to the growing number of
secreted cytotoxins that play a role in EHEC-intestinal interactions. Shiga toxin was recently observed to influence
intestinal colonization of mice by E. coli O157:H7, possibly
by modulating the surface expression of intimin coreceptors
(Robinson et al., 2006). Furthermore, an inhibitor of lymphocyte proliferation and cytokine synthesis (LifA, lymphostatin) influences intestinal colonisation in calves by EHEC
O5:H- and O111:H- (Stevens et al., 2002) and persistence and
the induction of colonic hyperplasia by Citrobacter rodentium
in mice (Klapproth et al., 2005). However, lifA mutations also
impair adherence (Stevens et al., 2002) and the contribution
of direct cytotoxicity to colonization is not fully understood.
Enterohaemolysin was also suggested to be important in
colonization of calves by EHEC O26:H- by STM and the gene
is highly conserved among EHEC (van Diemen et al., 2005).
It is possible that such cytotoxins act in concert to promote
intestinal colonization.
EspP influences adherence of E. coli O157:H7 to
bovine primary rectal epithelial cells
To assess the basis of attenuation, bovine primary rectal
epithelial cells were isolated for in vitro adherence assays.
(a)
1
2
3
(b)
250
100
75
50
1.E+06
Adjusted mean number of adhering
bacteria
150
Epithelial cells were obtained from the terminal rectum of
cattle c. 0–5 cm proximal to the rectal–anal junction. The
relevance of cells derived from this site is suggested by
the finding that colonization of cattle can be established by
the direct administration of E. coli O157:H7 to terminal
rectal mucosa (Sheng et al., 2004) in a manner dependent on
pO157 (Sheng et al., 2006). The cultured cells were polarized
and differentiated, expressed the epithelial cell-specific cytokeratins and resembled those seen previously with bovine
colonic primary epithelial cell cultures (Hoey et al., 2003).
To examine the impact of secreted EspP on adherence,
EspP were overexpressed in E. coli strain HB101 and the
protein from the supernatant was purified. The purity of the
protein preparation was confirmed by SDS-PAGE (Fig. 3a)
and the identity of the protein species verified by Western
blotting (data not shown). The purification method has
previously been used to prepare homogenous catalytically
active EspC from EPEC (Mellies et al., 2001; Navarro-Garcia
et al., 2004).
Mutant and parent strains were allowed to interact with
primary rectal epithelial cells in the presence or absence of
purified exogenous EspP. The mean adherence data indicated that the 85-170nalR DespP::kanR mutant was significantly (P o 0.001) impaired in its ability to adhere to bovine
rectal primary epithelial cells compared with the parent
strain (Fig. 3b). The cells showed no evidence on cytotoxicity or cytoskeletal changes (data not shown). The adherence defect of the mutant was rescued by addition of highly
1.E+05
1.E+04
37
1.E+03
25
Wildtype
Wildtype plus
EspP
Mutant
Mutant plus
EspP
Class of data
Fig. 3. Adherence of Escherichia coli strain 85-170nalR and its DespP::kanR mutant to bovine primary rectal epithelial cells in the presence or absence of
purified exogenous EspP. The purity of exogenous EspP was assessed by SDS-PAGE (a). Lane 1, standard molecular weight marker (kDa); lane 2, whole
cell lysate of strain HB101 (pB9-5espP) overproducing EspP; lane 3, purified EspP following ultrafiltration with a 100 kDa cut-off centricon. Adherence
data represent the mean from three independent assays (b).
FEMS Microbiol Lett xx (2007) 000–000
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Published by Blackwell Publishing Ltd. All rights reserved
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6
purified recombinant EspP, suggesting that the phenotype of
the mutant was due to loss of EspP rather than due to
second-site or polar effects. Adherence of the wild-type
strain was increased by the addition of exogenous EspP, but
not significantly so (P = 0.03).
Although the mechanism by which EspP affects intestinal
colonization and adherence to cultured cells is unknown, a
previous study indicated that it cleaves pepsin A and human
coagulation factor V (Brunder et al., 1997). Degradation of
factor V is a shared feature among SPATES from other E. coli
pathotypes (Dutta et al., 2002) and this has been suggested
to contribute to mucosal haemorrhage in humans (Brunder
et al., 1997). Some SPATEs possess mucinase activity (reviewed in Henderson et al., 2004); however, it has been
reported that EspP is unable to cleave bovine submaxilliary
mucus in vitro (Dutta et al., 2002). Type V-secreted proteins
typically possess a b-domain that remains anchored on the
bacterial outer membrane and a passenger domain that is
often cleaved and released in the milieu. Recently, it has been
reported that some autotransported proteins are capable of
mediating adhesion directly (Fink et al., 2003) or by acting
as scaffolds for bacterial adhesins (Veiga et al., 2003) and it
remains possible that surface-anchored EspP may act in this
way. The contribution of the secreted and surface-associated
portions of EspP and other EHEC cytotoxins in colonization
and pathogenesis merits further investigation.
Acknowledgements
The authors gratefully acknowledge the support of the
Department for the Environment, Food and Rural Affairs
(project no. OZ0707), the support from DEFRA VTRI
(project no. VT0102) and the Scottish Executive Environment and Rural Affairs Department (WP2.2-22050).
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