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Infection and Immunity, December 1999, p. 6710-6714, Vol. 67, No. 12
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Isogenic Lysogens of Diverse Shiga Toxin 2-Encoding
Bacteriophages Produce Markedly Different Amounts of Shiga
Toxin
Patrick L.
Wagner,
David
W. K.
Acheson, and
Matthew K.
Waldor*
Division of Geographic Medicine and
Infectious Diseases, New England Medical Center and Tufts
University School of Medicine, Boston, Massachusetts 02111
Received 16 August 1999/Returned for modification 9 September
1999/Accepted 17 September 1999
 |
ABSTRACT |
We produced isogenic Escherichia coli K-12 lysogens of
seven different Shiga toxin 2 (Stx2)-encoding bacteriophages derived from clinical Shiga toxin-producing E. coli (STEC) isolates
of serotypes O157:H7, O145, O111, and O83 to assess the variability among these phages and determine if there were phage-related
differences in toxin production. Phage genomic restriction fragment
length polymorphisms (RFLP) and superinfection resistance studies
revealed significant differences among these phages and allowed the
seven phages to be placed into five distinct groups. Experiments
revealed striking differences in spontaneous phage and toxin production that were correlated with the groupings derived from the RFLP and
resistance studies. These results suggest that the genotype of the Stx2
prophage can influence the level of phage release and toxin expression
by host strains and thus may be relevant to STEC pathogenesis.
| |
TEXT |
Escherichia coli O157:H7
and other Shiga toxin-producing E. coli (STEC) are emerging
pathogens responsible for both outbreaks and sporadic cases of diarrhea
(12). In addition to diarrhea, some patients develop
hemorrhagic colitis, hemolytic uremic syndrome, or thrombotic
thrombocytopenic purpura after exposure to STEC (9). These
severe clinical consequences of STEC infection are believed to be
caused by the activity of Shiga toxins 1 and 2 (Stx1 and Stx2),
although Stx2 appears to be more closely associated with these sequelae
(4, 17, 20). E. coli isolates of more than 60 serotypes have been found to produce Shiga toxins and to be associated
with human disease (2). The genes encoding the Shiga toxin A
and B subunits in several well-studied STEC isolates have been found to
reside on lambdoid prophages (11, 16), and STEC isolates
appear, as a rule, to be lysogens, converted to toxinogenicity by
bacteriophages. The role of the biology of these phages in host strain
virulence, beyond merely carrying the Shiga toxin genes to the STEC
strains during evolution, has been relatively unexplored. Recently,
however, it was demonstrated that a phage-encoded transcription factor
is able to activate Shiga toxin expression, suggesting a mechanism
whereby phage regulation might influence host strain virulence
(15). Furthermore, intraintestinal transmission of these
phages has been demonstrated to occur among E. coli strains
(3), and infectious Stx2-encoding phages were recently
detected in municipal sewage (14), emphasizing that Stx-encoding bacteriophages have retained the capacity to disseminate Shiga toxin genes among nontoxigenic E. coli and other
bacterial species as well.
Stx2-encoding bacteriophages may thus play a more active role in STEC
pathogenesis than simply acting as the carrier of toxin genes, and an
assessment of the level of biological diversity among these phages
could reveal variations in, for example, levels of toxin expression
relevant to the virulence of their hosts. Significant genomic
divergence among Stx1-encoding phages isolated from different E. coli serotypes has been demonstrated by analysis of phage genome
lengths and restriction fragment length polymorphisms (RFLP)
(25). Recent studies have revealed some variation within O157-derived Stx2-encoding phages (6, 22). In the present study, to assess the level of variation among Stx2-encoding phages derived from multiple STEC serotypes and its possible relevance to
toxin regulation, we produced isogenic lysogens of seven different Stx2-encoding phages. These seven lysogens were used in RFLP and superinfection resistance studies, which allowed their phages to be
placed into five distinct groups. Investigation of spontaneous phage
release and toxin production from these isogenic lysogens revealed
striking differences in both phage titer and toxin production that were
consistent with the RFLP and resistance groupings. These experiments
suggest that the genotypes of the Stx2 prophages can influence the
level of toxin expression by host strains and thus may be relevant to
STEC pathogenesis.
Construction of isogenic lysogens, RFLP analysis, and
superinfection resistance analysis.
We set out to examine the
diversity of Stx2-encoding phages derived from various clinical STEC
isolates of multiple serotypes and to investigate the possible
contribution of phage heterogeneity to the regulation of toxin
expression. The places and dates of isolation of the Stx2-encoding
clinical isolates used in this study are presented in Table
1. These clinical isolates were obtained
from stool samples of patients with either bloody diarrhea or hemolytic
uremic syndrome (1). In order to eliminate the contribution
of additional prophages or host factors in the diverse STEC clinical
isolates from which Stx2-encoding phages were isolated, we purified
Stx2-encoding phages from single plaques and subsequently lysogenized
an E. coli K-12 derivative, MC1000 (5), with the purified phage. We were successful in producing MC1000 lysogens of six
new Stx2-encoding phages, as well as phages 
, 933W (the prototype
Stx2-encoding phage), and H19B (the prototype Stx1-encoding phage.)
Phage genomic DNA was isolated from each of the seven Stx2-encoding
phages (including 933W) by use of a commercial kit (Qiagen,
Valencia,
Calif.) RFLP among the seven phage genomes were detected
by digestion
with the enzymes
AvaI,
BanI,
EcoRI,
and
EcoRV. The
marked polymorphism in fragment lengths seen
in Fig.
1 was used
to assign the seven
phages to five distinct RFLP groups. Lysogens
of each phage were tested
for resistance to superinfection by
the other phages. This was done by
spotting a sample of each of
the seven phages on lawns of each lysogen
and noting the presence
or absence of lysis. Each lysogen was found to
be resistant to
infection by its own phage and susceptible to phages
and H19B
(Table
2). Furthermore,
resistance was observed within but not
among the five groups assigned
by RFLP analysis. Thus, phages
933W and 10 shared a RFLP pattern and
were mutually resistant
to superinfection; phages 2 and 3 constituted a
similar pair,
while each of the remaining phages (phages 14, 16, and
19) constituted
its own group. Our finding of five distinct groups
among seven
isolated phages implies that a high level of diversity
exists
among Stx2-encoding phages, especially considering the fact that
our methods selected for phages sufficiently similar to lysogenize
a
common host. The relative conservation of the Shiga toxin gene
sequences (as indicated by the fact that Stx2A sequences from
all seven
phages could be amplified with the same PCR primers)
in the context of
such diversity suggests that these genes were
acquired relatively
recently by this group of phages. This likely
reflects significant
ongoing recombination within the lambdoid
phage gene pool (reviewed in
reference
10).
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FIG. 1.
RFLP in the Stx2-encoding genomes. Phage DNA was
digested with the indicated restriction enzymes and electrophoresed in
an agarose gel containing ethidium bromide. In the gel, the order of
the digested phage DNA for each restriction enzyme is the same:
 933W, 10, 2, 3, 14, 16, and 19 (from left to
right). M, HindIII-digested phage DNA and
HaeIII-digested X174 DNA markers.
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|
Differences between Stx2-encoding phages from serotype O157 STEC
isolates have been reported (
6,
22). Our results extend
these findings to include Stx2-encoding phages derived from STEC
of
various serotypes, including O157, O145, O111, and O83. We
found no
evidence for a relationship between the RFLP and resistance
properties
of the phages and the serotype of the STEC strain from
which they were
isolated. Specifically, phages 933W and 10 were
indistinguishable by
RFLP and resistance analysis yet were isolated
from strains of
serotypes O157 and O145, respectively; conversely,
phages 1, 3, and 14
all derived from O157 strains
were readily
distinguished by RFLP
and resistance studies. Among the various
STEC serogroups, O157 strains
have been shown to be closely related
and are believed to share a
common ancestry (
24). It has been
proposed that an early
progenitor of the O157:H7 STEC complex
was converted to toxinogenicity
by an Stx2-encoding phage (
23).
Our findings of diverse Stx2
phages in different O157:H7 strains,
taken together with the recent
finding of two different O157-derived
Stx2-encoding phages in Japan
(
22), suggests that Stx2 phage
infection and lysogenic
conversion of O157:H7 may have occurred
more recently in STEC evolution
than was previously appreciated
and almost certainly rules out a single
conversion event early
in O157:H7
evolution.
Toxin and phage production by MC1000 lysogens and clinical
isolates.
The significant genomic variation revealed by RFLP
analysis suggested the possibility that differences in toxin production among the lysogens might also exist. Therefore, we examined amounts of
spontaneous phage and toxin produced by the MC1000 lysogens. Phage
titers were obtained by infecting lawns of MC1000, and total Stx2 from
sonicated cultures was measured by a previously described enzyme-linked
immunosorbent assay (7). Figure 2A and
B presents the striking differences
observed among the seven MC1000 strains in both phage and toxin
production measured during mid-logarithmic growth phase. Large
differences were observed among but not within the groups defined by
the RFLP and resistance studies. Furthermore, some correlation between
the amounts of toxin and phage produced is evident from the observation
that the order of the seven lysogens ranked by phage titer is nearly
identical to that ranked by toxin production. When normalized to total
culture protein concentration, differences in toxin production by these
isogenic strains varied by as much as 200-fold at 3 h after
subculture (Fig. 2A) and exceeded 800-fold after overnight growth (data
not shown). Differences in phage titer reached 105-fold at
3 h (Fig. 2B). Since our MC1000 lysogens differ only in their Stx2
prophage genotype, our finding of significant variation in toxin
production indicates that phage genotype has an unmistakable influence
on toxin expression by the lysogens. To the list of phenotypic
variations among Shiga toxin-encoding bacteriophages, then, we add the
potentially clinically germane characteristic of toxin expression level
in a common host strain.
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FIG. 2.
(A) Toxin production in the presence (black bars) or
absence (gray bars) of mitomycin C (0.5 µg/ml) by MC1000 lysogens of
the phages indicated. (B) Phage production in the presence (black bars)
or absence (gray bars) of mitomycin C by MC1000 lysogens of the phages
indicated. (C) Toxin production in the absence of mitomycin C by the
clinical isolates from which the indicated phages were obtained. *,
O157:H7 clinical isolates. Mean values are shown along with standard
deviations derived from three independent trials.
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|
In order to determine whether the variation we observed in toxin
production among the MC1000 lysogens also exists among the
clinical
STEC isolates from which the Stx2 phages were isolated,
we measured the
Stx2 concentration in cultures of each clinical
isolate grown
overnight, as shown in Fig.
2C. Corrected for total
protein
concentration, uninduced Stx2 production varied by up
to 480-fold. No
relationship was found between toxin production
by a given clinical
isolate and toxin production by the MC1000
lysogen of the phage derived
from that clinical isolate. For instance,
Fig.
2C shows a large
difference in toxin production by the clinical
hosts of phages 933W and
10, despite the fact that MC1000 lysogens
of these phages make similar
amounts of toxin. A relationship
between serotype and toxin production
is, however, suggested by
the result that the four O157:H7 strains
produced more toxin than
the non-O157 strains (Fig.
2C). Unlike the
single plaque-purified
MC1000 lysogens, the clinical isolates are
generally lysogens
of multiple prophages. Our inability to distinguish
plaques from
different phages made an assessment of specific phage
release
from clinical isolates
impractical.
Mitomycin C was added to cultures of each MC1000 lysogen to study the
effects of exogenous induction on phage release and
toxin production.
After 3 h (by which time lysis was visually
apparent in each
mitomycin-treated culture), we determined the
phage titer and Stx2
concentration in each lysate. As shown in
Fig.
2, mitomycin C induction
substantially reduced the differences
in the phage and toxin production
described above among the cultures
of uninduced lysogens. Thus, the
Stx2 concentration, when corrected
for total protein concentration,
varied by no more than 6-fold
(Fig.
2A), while variation in the phage
titer was reduced to less
than 200-fold (Fig.
2B). Among the seven
lysogens, the augmentation
of toxin and phage production following
mitomycin C treatment
was most pronounced in those strains which had
the lowest titers
of toxin and phage in the absence of mitomycin C
treatment (Fig.
2). Mitomycin C induction of clinical isolates was not
carried
out because of the presence of multiple prophage repression
systems
in each strain. One possible explanation for our findings of
variation
in phage and toxin production is that the different phages,
upon
lysogenization of the MC1000 host, could in some way damage the
capacity of the host to generate phage or toxin. The equilibration
of
phage and toxin production by mitomycin C illustrates that
the various
strains retain a comparable capacity for phage and
toxin release and
suggests that in certain strains phage and toxin
expression are held in
check by some factor that is removed upon
treatment with mitomycin
C.
The genotypic variation among the bacteriophages which accounts for the
diverse levels of toxin production by the uninduced
MC1000 lysogens is
not known. While a variety of mechanisms for
phage control of toxin
production are conceivable and more than
one may be important, the most
parsimonious explanation involves
differences in prophage repression.
The
prophage encodes a repressor,
cI, which is inactivated by
RecA-dependent autocleavage as part
of the SOS response. This cleavage
is the critical molecular event
associated with the switch from the
lysogenic to the lytic phase
of the temperate phage life cycle
(
19). Previous work has established
that SOS-inducing
agents, such as mitomycin C, increase both phage
titer and toxin
production from particular Shiga toxin phage lysogens
(
13).
Furthermore, the Shiga toxin genes in 933W and H19B have
been located
in the late region of each prophage (
15,
18).
Following
induction and repressor cleavage in lambdoid prophages,
phage-encoded Q
antiterminators (reviewed in reference
8) act
at DNA
qut sites located between promoters and terminators to
modify passing RNA polymerase complexes in a way that renders
them
insusceptible to termination. In this way, the late genes,
held
quiescent in lysogenic phase by the presence of a strong
stem-loop
terminator, tR', just downstream of the late promoter,
can be expressed
following the lytic switch when the Q antiterminator
becomes available.
Neely and Friedman (
15) showed that overexpression
of the Q
antiterminator of 933W enhances toxin expression from
its prophage in
the K-12 derivative C600, as well as expression
of downstream lysis
genes. This suggests that lysis genes and
the toxin subunit genes are
cotranscribed from the late phage
promoter pR', a possibility that is
particularly attractive since
no specific Stx2 secretory mechanism has
been described, and that
phage-mediated lysis may be a mechanism for
toxin release. This
body of evidence suggests that the regulation of
toxin production
is dependent on phage development, i.e., that toxin
production
is part of the coordinated expression of late genes at the
appropriate
time in the phage lytic cycle following induction. This
represents
a departure from the conventional view that Stx2-converting
phages
strictly serve as vectors for horizontal transfer of toxin genes
among STEC strains (
21).
Our observations add to this model in two ways. First, we observed the
trend among seven Stx2-encoding phages that phage particle
production
and toxin production vary together, which strengthens
the idea that
they are coordinately regulated. Second, although
we have not strictly
shown that our phages exhibit immunity in
the lambdoid sense (i.e.,
immunity based upon common repressors
rather than, for example, surface
exclusion), our finding that
various phage groups are susceptible to
superinfection by each
other indicates that they differ in their
repressor regions. Thus,
in cases where significant differences in
phage and toxin production
exist between two lysogens, we know that, at
the least, they have
different prophage repression systems. Conversely,
in MC1000 lysogen
pairs that appear to be homoimmune (i.e., to share
functionally
identical repressors), we observed no significant
difference in
toxin production. It is possible that some Stx2-encoding
phages
have weaker repressors than others, weaker in this context
meaning
more susceptible to RecA-mediated cleavage in MC1000. We
speculate
that such repressor differences are the prophage genotypes
most
likely responsible for the observed differences in toxin and phage
production in the MC1000
background.
Other host- or bacteriophage-encoded factors besides or in addition to
relative repressor strength may be influencing toxin
and phage
production as well. For instance, different phage integration
sites
could at least partially explain the differences in toxin
expression by
the MC1000 lysogens. Since most lambdoid phages
integrate site
specifically via phage-encoded recombinases, this
again would be an
example of a phage genotype directing the level
of toxin expression.
Alternatively, differences in expression
and activity of lysis genes
could influence the stability of lysogeny.
Despite these other
possibilities, the observation that variation
among the MC1000 lysogens
was substantially blunted by mitomycin
C
an agent known to bring about
RecA-mediated repressor cleavage
strongly
implies that repressor
differences explain toxin and phage production
differences.
No relationship was found between the amount of toxin produced by the
MC1000 lysogens and that produced by the clinical isolates
from which
each MC1000 lysogen's phage was derived. In most instances,
the
uninduced clinical isolates produced much less toxin than
their
uninduced MC1000 counterparts at comparable points in mid-logarithmic
phase (data not shown), making it necessary to measure toxin release
by
the clinical isolates after overnight growth (Fig.
2C) instead
of after
3 h of growth, as in the MC1000 strains (Fig.
2A). This
may
reflect the evolution of the prophage relationship with its
natural
host, in which selection would presumably favor repressor
systems which
act to more stably maintain lysogeny, with less
lytic switching and
therefore less toxin production. Further complicating
matters in the
clinical isolates is the fact that STEC isolates
generally contain
numerous prophages. Frequently, Stx1- and Stx2-encoding
prophages are
found in the same strain, along with non-Shiga toxin-encoding
prophages, and in fact at least one of our clinical isolates
contains
two heteroimmune Stx2-encoding phages. With each additional
prophage
repression system present, the control of Stx2 production may
move further away from the lytic switch of the encoding prophage
and
more toward a complex matrix of interactions between phage
repressors
and host factors like RecA. Clinical intervention to
prevent the lytic
switch by prophages in STEC strains would represent
a novel
strategy
based upon the underlying phage biology
to avoid
the
clinical sequelae of STEC
infection.
| |
ACKNOWLEDGMENTS |
We are grateful to B. Davis, F. Boyd, and A. Kane for critical
reading of this manuscript and A. Kane and the NEMC GRASP Digestive Disease Center for preparing the microbiologic media for our studies.
This work was supported by grants AI-42347 to M.K.W., AI-39067 to
D.W.K.A. and P30DK-34928 for the NEMC GRASP Digestive Center. M.K.W. is
a Pew Scholar in the Biomedical Sciences and a Tupper Research Fellow.
P.L.W. is supported by a Harvard Medical School Student Research
Fellowship and a Howard Hughes Medical Institute Research Training
Fellowship for Medical Students.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Geographic Medicine and Infectious Diseases, New England Medical Center no. 041, 750 Washington St., Boston, MA 02111. Phone: (617) 636-7618. Fax: (617) 636-5292. E-mail:
matthew.waldor{at}es.nemc.org.
Editor:
J. T. Barbieri
| |
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Infection and Immunity, December 1999, p. 6710-6714, Vol. 67, No. 12
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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