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Infection and Immunity, August 2001, p. 5107-5114, Vol. 69, No. 8
ZAP Laboratories, Department of Veterinary
Pathology, University of Edinburgh, Edinburgh,1
and Department of Medical Microbiology, University of Aberdeen,
Aberdeen,2 United Kingdom, and Institut
für Medizinische Mikrobiologie,
Justus-Leibig-Universität, Giessen,
Germany3
Received 27 November 2000/Returned for modification 28 February
2001/Accepted 3 May 2001
Ongoing extensive epidemiological studies of verotoxin-carrying
Escherichia coli O157
(stx+
eae+) have shown this
bacterial pathogen to be common in cattle herds in the United States
and the United Kingdom. However, the incidence of disease in humans due
to this pathogen is still very low. This study set out to investigate
if there is a difference between strains isolated from human disease
cases and those isolated from asymptomatic cattle which would account
for the low disease incidence of such a ubiquitous organism. The work
presented here has compared human disease strains from both sporadic
and outbreak cases with a cross-section, as defined by pulsed-field gel
electrophoresis, of E. coli O157 strains from cattle.
Human (n = 22) and bovine (n = 31) strains were genotyped for carriage of the genes for Shiga-like
toxin types 1, 2, and 2c; E. coli secreted protein genes
espA, espB, and espP; the enterohemolysin
gene; eae (intimin); ast
(enteroaggregative E. coli stable toxin [EAST]); and
genes for common E. coli adhesins. Strains were
also phenotyped for hemolysin, EspP, Tir, and EspD expression as well
as production of actin and cytoskeletal rearrangement associated
with attaching and effacing (A/E) lesions on HeLa cells. The genotyping
confirmed that there was little difference between the two groups,
including carriage of stx2 and
stx2c, which was similar in both sets.
ast alleles were confirmed to all contain mutations that
would prevent EAST expression. espP mutations were found
only in cattle strains (5 of 30). Clear differences were observed in
the expression of locus of enterocyte effacement (LEE)-encoded factors
between strains and in different media. EspD, as an indicator of LEE4
(espA, -B, and -D) expression, and Tir
levels in supernatants were measured. Virtually all strains from both
sources could produce EspD in Luria-Bertani broth, although at very
different levels. Standard trichloroacetic acid precipitation of
secreted proteins from tissue culture medium produced detectable levels
of EspD from the majority of strains of human origin (15 of 20)
compared with only a few (4 of 20) bovine strains
(P < 0.001), which is indicative of much higher
levels of protein secretion from the human strains. Addition of bovine
serum albumin carrier protein before precipitation and enhanced
detection techniques confirmed that EspD could be detected after growth
in tissue culture medium for all strains, but levels from strains
of human origin were on average 90-fold higher than those from strains
of bovine origin. In general, levels of secretion also correlated
with ability to form A/E lesions on HeLa cells, with only the
high-level protein secretors in tissue culture medium exhibiting a
localized adherence phenotype. This research shows significant
differences between human- and bovine-derived E. coli O157 (stx+
eae+) strains and their production of
certain LEE-encoded virulence factors. These data support the recent
finding of Kim et al. (J. Kim, J. Nietfeldt, and A. K. Benson,
Proc. Natl. Acad. Sci. USA 96:13288-13293, 1999) proposing
different E. coli O157 lineages in cattle and humans and
extend the differential to the regulation of virulence factors.
Potentially only a subset of E. coli O157 isolates
(stx+
eae+) in cattle may be capable of
causing severe disease in humans.
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.8.5107-5114.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Differences in Levels of Secreted Locus of
Enterocyte Effacement Proteins between Human Disease-Associated and
Bovine Escherichia coli O157
*
Corresponding author. Mailing address: ZAP Laboratory,
Department of Veterinary Pathology, Teviot Place, Edinburgh EH8 9AG, United Kingdom. Phone: 0131-651-1342. Fax: 0131-650-6531. E-mail: dgally{at}ed.ac.uk.
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