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Infection and Immunity, November 1999, p. 5898-5905, Vol. 67, No. 11
Division of Geographic Medicine and
Infectious Diseases, Tufts-New England Medical Center and Tufts
University School of Medicine, Boston, Massachusetts 02111
Received 17 May 1999/Returned for modification 14 July
1999/Accepted 10 August 1999
Horizontal transfer of genes encoding virulence factors has played
a central role in the evolution of many pathogenic bacteria. The
unexpected discovery that the genes encoding cholera toxin (ctxAB), the main cause of the profuse secretory diarrhea
characteristic of cholera, are encoded on a novel filamentous phage
named CTX Vibrio cholerae is
unusual among enteric pathogens both for its tendency to cause
explosive outbreaks and for its predilection for pandemic spread.
V. cholerae is the prototypical noninvasive enteric pathogen
which is spread by the ingestion of contaminated food and water. Of the
more than 150 serogroups that exist, until recently only the O1
serogroup was associated with epidemic cholera. The O1 serogroup is
divided into two distinct biotypes, designated classical and El Tor.
The first six pandemics of cholera are believed to have been caused by
the classical biotype and the ongoing seventh pandemic, which began in
1961, is caused by the El Tor biotype (6). Remarkably, in
1992 a new epidemic serogroup, O139 Bengal, emerged and
temporarily replaced the predominant O1 serogroup (1, 9,
35).
Most of the major symptoms caused by V. cholerae infection
result from the production of cholera toxin (CT) in the small intestine after colonization (39). Recently, CT was found to be
encoded in the genome of an unusual lysogenic filamentous phage, named CTX The CTX
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Alternative Mechanism of Cholera Toxin Acquisition by
Vibrio cholerae: Generalized Transduction of CTX
by
Bacteriophage CP-T1
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, has resulted in a renewed interest in the potential
mechanisms of transfer of virulence genes among Vibrio
cholerae. We describe here an alternative mechanism of cholera
toxin gene transfer into nontoxigenic V. cholerae
isolates, including strains that lack both the CTX receptor, the
toxin coregulated pilus (TCP), and attRS, the chromosomal
attachment site for CTX integration. A temperature-sensitive mutant
of the V. cholerae generalized transducing bacteriophage CP-T1 (CP-T1ts) was used to transfer a genetically marked derivative of the CTX prophage into four nontoxigenic
V. cholerae strains, including two V. cholerae
vaccine strains. We demonstrate that CTX transduced by CP-T1ts can
replicate and integrate into these nontoxigenic V. cholerae
strains with high efficiency. In fact, CP-T1ts transduces the CTX
prophage preferentially when compared with other chromosomal
markers. These results reveal a potential mechanism by which
CTX+ V. cholerae strains that lack the
TCP receptor may have arisen. Finally, these findings indicate an
additional pathway for reversion of live-attenuated V. cholerae vaccine strains.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(45, 47). The receptor for CTX is thought to be a
type IV pilus, the toxin coregulated pilus (TCP), since V. cholerae cells that do not express TCP were found to be resistant
to CTX infection (40, 42, 45).
genome consists of a 4.6-kb core region and a 2.4-kb
repeated sequence (RS2) region. The 4.6-kb core region encodes the
cholera toxin A and B subunits (ctxAB) and the
psh, cep, orfU, ace, and
zot genes which are required for phage morphogenesis and
secretion. The 2.4-kb RS2 region encodes the rstR,
rstA, and rstB genes required for repression,
replication, and site-specific integration of CTX (47)
(Fig. 1).
View larger version (13K):
[in a new window]
FIG. 1.
Chromosomal arrangement of TLC, RS1, CTX , and RTX in
the V. cholerae chromosome. The open reading frames are
shown as arrows. The black triangles represent attRS
sequences. Vertical lines indicate restriction enzyme sites. Horizontal
bars show the five DNA probes used in this study.
After infection of classical V. cholerae strains and El Tor
strains lacking a CTX integration site, CTX can replicate as a
plasmid. This plasmid form of CTX was designated the phage replicative form (RF), since cells harboring the plasmid produce large
amounts of viral particles. However, upon infection of most El Tor
strains, CTX integrates into the chromosome at a unique attachment
site (attRS) to form a lysogen rather than maintaining the
RF (33, 45, 47). Often, an RS1 sequence is found immediately adjacent to the CTX prophage on the V. cholerae chromosome
(Fig. 1). RS1 is closely related to the RS2 region of the CTX genome but contains an additional open reading frame of unknown function, rstC. The function and mechanism of transfer of RS1 is
unknown. Upstream of the CTX prophage is an integrated 4.7-kb plasmid
named pTLC (toxin-linked cryptic element) (36). Located 3'
of the CTX prophage is a large RTX toxin gene cluster (27)
(Fig. 1).
The genes encoding the biosynthesis of TCP, the CTX receptor, reside
on a pathogenicity island known as the TCP island or vibrio
pathogenicity island (VPI) (22, 24). Recently, Karaolis et
al. have proposed that the VPI corresponds to the genome of another
lysogenic filamentous bacteriophage, VPI (23). Since TCP
is required for CTX infection of V. cholerae, it was
proposed that there were two critical sequential steps in the evolution of pathogenic V. cholerae (14, 15, 30, 45, 46).
First, strains had to acquire the TCP island (presumably via infection with VPI) and, second, having acquired the CTX receptor (as well
as ToxT, a VPI-encoded transcription factor critical in expression of
ctxAB), these TCP+ strains were then infected
with and lysogenized by CTX. Studies with the suckling mouse cholera
model revealed that CTX infection of nontoxigenic strains is
particularly efficient within the intestine, an environment known to
induce TCP expression, suggesting that the host environment may be the
site where TCP+ mutant ctxAB strains acquire
CTX (25, 45). In fact, in a recent extensive strain
survey, Faruque et al. (14) identified TCP+
CT+ strains and TCP+ CT
strains but found no TCP CT+ strains,
suggesting that CTX was acquired subsequent to the acquisition of
TCP. However, the two-step model of the sequential evolution of
pathogenic V. cholerae is called into question by several
V. cholerae O1 and non-O1 isolates that have been recently described that lack TCP but contain CTX sequences (18,
38). These strains indicate that acquisition of CTX and TCP
may be independent of one another.
In the current study we investigate whether ctxAB is
transmissible by means other than via CTX. We found that
CP-T1ts, a V. cholerae generalized transducing phage
(32), transfers the entire CTX genome at a high
frequency. CP-T1ts-transduced CTX can integrate into alternative
attachment sites, and TCP is not required for CP-T1ts-mediated
acquisition of CTX. These findings cast doubt on the assumption that
CTX is the sole mediator of horizontal transfer of CT genes and that
transfer occurs primarily within the host environment.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacterial strains.
The bacterial strains used in this study
are shown in Table 1. Bacterial strains
were stored at 
70°C in Luria-Bertani (LB) broth containing 20%
glycerol. Antibiotics were used at the following concentrations:
kanamycin (Kn), 50 µg/ml; streptomycin (Sm), 200 µg/ml; and
tetracycline (Tc), 1 µg/ml.
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CP-T1 transduction. We performed experiments with a temperature-sensitive (ts) mutant of the V. cholerae transducing phage, CP-T1 (32), to determine whether ctxAB was transmissible by transduction to nontoxigenic V. cholerae O1 recipient strains. To detect transduction of ctxAB, a CP-T1ts phage was cultured and purified from strain SM115 (19), which contains Kn-marked ctxAB, to produce a high-titer lysate as follows. Serial dilutions of CP-T1ts were added to 20-µl aliquots of SM115 overnight cultures, mixed, and incubated for 10 min to allow CP-T1 infection of strain SM115. Infected cells were then added to 5.5 ml of top agar, mixed, and spread on LB agar plates. The LB plates were incubated overnight, and 5 ml of LB broth was added to those plates with confluent plaques. The LB broth and top agar were recovered in a large culture tube, mixed, and left at 4°C overnight to allow phage diffusion from the top agar. The top agar-phage mixture was centrifuged for 1 min at top speed in a bench-top centrifuge; the supernatant was then recovered and further centrifuged for 30 s to remove agar. The supernatant was then centrifuged for 2 h at 4°C at top speed in an Eppendorf microcentrifuge to concentrate the phage, followed by immediate aspiration of all but 60 µl of the supernatant, in which the phage was resuspended. A drop of chloroform was added to kill any remaining bacterial cells, and the high-titer lysate (1010 to 1011 PFU/ml) was stored at 4°C.
Transduction experiments were carried out with 20 µl of this high-titer lysate treated with 5,000 µJ of UV light in a Stratagene Stratalinker, which inactivates 90 to 99% of the phage. UV-treated phage were added to 100 µl of late-log-phase recipient cells, mixed briefly, and then incubated for 6 min to allow CP-T1 infection of recipient cells. To this mixture, 1 ml of LB medium was added, and it was then incubated at 39°C for 2 h with shaking. Next, cells were pelleted and resuspended in 100 µl of LB broth. Aliquots of 10 and 90 µl were plated on Kn-containing selection plates and incubated overnight at 39°C, which prohibits CP-T1ts replication. To determine relative transfer efficiency of CTX-Kn by CP-T1, a
second chromosomal marker was transduced from SM115 for comparison purposes. This was accomplished by initially infecting V. cholerae V66 (8), which contains a Tc-marked
lacZ gene, with CP-T1. This lysate was then used to
transduce SM115 to tetracycline resistance. The resulting strain,
SM115-Tc, then contains a Tc-marked lacZ and a Kn-marked
CTX.
Molecular analyses.
Total genomic DNA from each bacterial
isolate was extracted by using the G-nome DNA isolation kit from Bio
101 (Vista, Calif.). DNA was digested with several restriction enzymes,
and the fragments were separated by electrophoresis in 0.6% agarose.
The fragments were transferred to nylon membranes for hybridization as
previously described (7). Primers for PCR amplification were
designed from published DNA sequences (Table
2). PCR products were purified by using
the Qiaquick PCR purification kit (Qiagen, Valencia, Calif.). A total
of five DNA fragments were produced by PCR amplification (Table 2 and
Fig. 1) for use as nonradioactive probes by labeling with
fluorescein-conjugated nucleotides and, after hybridization, were
detected by the Amersham ECL System (Arlington Heights, Ill.). The
attRS probe was a PCR-amplified 328-bp fragment derived from strain 2740-80 (33) by using previously described primers
(33). The rstA, rstC, core, and TLC probes were made by PCR
amplification from strain SM115.
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particles. To accomplish this, supernatants from
mid-log-phase Knr transductants were filtered sterilized
and then used to transduce agglutinated (TCP+) classical
strain 0395 to Knr (31) according to an
established protocol (45). To determine whether
transductants derived from the transduction experiments contained
plasmid DNA, Qiagen plasmid spin kits were used to isolate DNA from 1 ml of overnight cultures of Knr transductants. These
plasmid DNA preparations were digested with SphI, separated
on agarose gels, and stained with ethidium bromide to visualize the
~7-kb plasmid CTX DNA band.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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CP-T1ts transduction of CTX.
The V. cholerae
phage CP-T1 has been shown to transduce a number of V. cholerae loci (21, 32). We used CP-T1ts, a
temperature-sensitive mutant of CP-T1, to study whether
ctxAB could be mobilized from an El Tor CTX lysogen to a
number of CTX O1 strains. To facilitate detection of
ctxAB transduction, CP-T1 lysates were made on strain SM115
(19). This El Tor strain contains tandemly arranged CTX
prophages that each contain an insertion of a Knr marker in
place of most of ctxAB
(
ctxABN4::Kn). Thus, kanamycin could
be used to select for CP-T1 transduction of ctxAB. In cases where Knr transductants were isolated, we investigated
whether the transductants contained the entire CTX DNA sequence as
well as chromosomal DNA sequences that flank the CTX prophage.
RS1 El Tor V. cholerae strains for transduction to Knr. We found
that lysates of SM115 could transduce Knr to all four
recipient strains even though not all strains possessed both TCP and
attRS. For example, strain 468-83, an El Tor strain that
lacks both attRS and TCP, the CTX receptor
(36), could be transduced to Knr at 1.5 × 106 transductants per PFU, indicating efficient
transduction of the ctxABN4::Kn
marker by CP-T1ts. Similarly, there was a high frequency of
Knr transductant colonies of strain 2740-80, an
attRS+ U.S. Gulf Coast isolate (33),
and of Bah-2 and Bah-3, two live-attenuated El Tor vaccine constructs,
which contain the "attRS" deletion of the entire CTX
prophage, RS1, and attRS from strain E7946 (33). Thus, CP-T1ts could efficiently transduce the
ctxABN4::Kn marker into a variety of
El Tor strains.
To examine whether CTX genes other than ctxAB had been
transferred, we tested whether infectious CTX-Kn virions were
produced by the Knr transductants of 468-83, 2740-80, Bah-2, and Bah-3. Filtered supernatants of individual transductants
were incubated with classical strain O395, a strain that is very
proficient in uptake of CTX in vitro (45), and the growth
of Kn-resistant O395 was then assayed. Cell-free supernatants from
468-83Kn, 2740-80Kn, Bah-2Kn, and Bah-3Kn transductants were found to
be capable of transducing O395 to Knr. The titer of Kn
transducing particles in supernatants derived from these four strains
did not differ by more than 1 order of magnitude (from 2 × 105 to 2 × 106 transducing particles/ml
of supernatant). Therefore, the Knr transductants of
468-83, 2740-80, Bah-2, and Bah-3 all produce infectious CTX-Kn
virions, indicating that the entire CTX genome, not just the
ctxABN4::Kn allele, was transduced
by CP-T1ts. Thus, CP-T1ts enables the horizontal transfer of CTX
genes to V. cholerae strains that lack the CTX receptor
TCP as well as to TCP+ strains.
Determination of the structural state of the CP-T1ts-transduced
CTX-Kn.
To determine the form in which CTX-Kn was maintained
in the Knr transductants, we analyzed plasmid DNA and
chromosomal DNA from a single Knr transductant of each of
the recipient strains. First, plasmid DNA prepared from one of each of
the Knr transductants, 468-84Kn, 2740-80Kn, Bah-2Kn, and
Bah-3Kn, was examined for the presence of the 6.9-kb plasmid RF of
CTX-Kn, pCTX-Kn. Plasmid DNA was isolated from strains 468-83Kn,
2740-80Kn, Bah-2Kn, Bah-3Kn, and O395 (pCTX-Kn) digested with
SphI, which cuts once within pCTX-Kn, and subsequently
probed with a core region probe (Fig. 1) in a Southern blot. All four
of the transductants yielded hybridizing DNA which migrated at 6.9 kb,
the size of the CTX RF; thus, each of the transductants contained
recoverable pCTX-Kn (Fig. 2).
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genome was also
integrated into the chromosome of 468-83Kn, 2740-80Kn, Bah-2Kn, and
Bah-3Kn, in addition to being present as a plasmid. For these studies,
Southern hybridization analyses were carried out with chromosomal DNA
prepared from each of the Knr transductants, as well as
with DNA derived from the original strains. These DNA preparations were
digested with restriction enzymes that cut once in the CTX genome.
Thus, strain 468-83Kn chromosomal DNA was digested with
XbaI, which cuts within ctxA at the site where
the Knr gene is inserted. Hybridization with a CTX core
region probe (Fig. 1 and Table 2) yielded a banding pattern identical
to that of the donor strain SM115 (Fig.
3). Two hybridization bands were obtained: an ~10-kb band corresponding to a CTX prophage and an adjacent copy of RS1 and a >10-kb band indicating the left junction fragment of the most 5' copy of RS1 (Fig. 3). This finding suggested that at least two copies of CTX and at least two copies of RS1 had
been transferred by CP-T1ts. To confirm the presence of the copies of
RS1, 468-83Kn DNA was digested with BglII, which cuts once
within the RS sequence, and then probed with an RS1-specific probe,
rstC (Fig. 4). This also resulted in a
banding pattern similar to that for SM115 strain (Fig. 4). Two bands
were visualized (Fig. 4), an ~2.7-kb band that represents the two RS1
sequences 5' of the two CTX prophages and an ~4-kb 3' junction
fragment corresponding to an RS1 3' of the CTX prophages (Fig. 4 and
5). Thus, strain 468-83Kn has two copies
of RS1 arranged similar to those of strain SM115.
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by CP-T1ts (not shown), as was
suggested by the equivalent sizes of the left junction fragments in
Fig. 3. To further confirm the tight linkage between TLC and CTX and
to assess whether RTX genes and CTX genes are linked in strain
468-83Kn, PCR analyses were performed. TLC, RTX, and CTX primers
were used to amplify the 5' and 3' flanking regions of the transduced
RS1 and CTX copies. PCR analysis confirmed the presence of a TLC
element located 5' of RS1A and an RTX element located 3' of RS1C in
strain 468-83Kn (data not shown) (Fig. 5). These results demonstrate
that not only CTX but also flanking regions from donor strain SM115
have been transduced by CP-T1ts into 468-83, suggesting that
nontoxigenic strains may obtain relatively large chromosomal regions
encoding a number of virulence genes in a single transfer event. Also, since strain 468-83 lacks the chromosomal attachment site for CTX,
it is likely that the large CP-T1ts-transduced fragment from donor
strain SM115 integrated into the 468-83 chromosome by homologous or
illegitimate recombination, rather than by CTX-mediated site-specific recombination.
We performed a similar analysis of the Knr transductants of
strain 2740-80, a nontoxigenic attRS+ El Tor
isolate from the U.S. Gulf Coast (33). Southern
hybridization analysis of DNA prepared from 2740-80Kn transductants
digested with XbaI and probed with a core region probe
revealed only a single hybridizing band of similar size to the 5'
junction fragments detected in SM115 and 468-83Kn, suggesting a single
integration of CTX (Fig. 3). Hybridization with the RS1-specific
probe rstC revealed a banding pattern similar to that of 468-83Kn with
two hybridization species. The ~2.7-kb band corresponds to an
integrated copy of RS1 located 5' of the integrated CTX (RS1A) and
the ~4 kb corresponds to an RS1 integrated located 3' of the CTX
prophage (RS1B) (Fig. 4 and 5). In strain 2740-80Kn, TLC and RTX
sequences were found 5' and 3', respectively, of the integrated CTX
by both Southern blot and PCR analyses (Fig. 5 and data not shown). The
reason why only a single RS1-CTX-RS1 was transduced from SM115 into
2740-80 and the entire RS1-CTX-RS1-CTX-RS1 and flanking DNA was
transduced into 468-83 is not clear. Since 2740-80 possesses attRS and 468-83 lacks attRS, this discrepancy
may reflect the possibility that RS1-CTX can integrate as a single
genetic element at attRS, perhaps via an RS-encoded
site-specific integration mechanism.
Bah-2 is a live-attenuated vaccine strain derived from El Tor strain
E7946 (33). Bah-2 contains a deletion of the two CTX prophages, the three RS1 elements, the attRS attachment
sites, and some flanking DNA from E7946 (Table 1). Perhaps because of this deletion CTX-Kn was integrated into the Knr
transductants of Bah-2 in a manner that differed from that of the
468-83Kn and 2740-80Kn transductants. In Southern analyses of
XbaI-digested DNA prepared from Bah-2Kn transductants, the core region probe detected two bands (Fig. 3), indicating that there
are two copies of CTX tandemly integrated into the Bah-2 chromosome.
The >10-kb band represents the 5' junction fragment of CTX in the
chromosome, and the ~6-kb band corresponds to tandem integrated
copies of CTX lacking an intervening RS1 (Fig. 3). We believe that
this ~6-kb band represents tandemly arranged CTX prophages (and not a
CTX plasmid form) because the intensity of this band in this blot is
at least equal to the intensity of the plasmid band detected in a
Southern blot of plasmid DNA derived from more than 100 times the
number of cells that the chromosomal DNA used in this blot was derived
from. The RS1-specific probe rstC gave no hybridization bands
indicating the absence of RS1 sequences in these transductants (Fig.
4). PCR analyses of the 5' and 3' flanking regions of the tandemly
integrated CTX prophages failed to reveal TLC and RTX sequences linked
to these CTX prophages (Fig. 5). These results indicate that transfer
and integration of CTX in this vaccine strain is not dependent on
attRS, RS1, TLC, or RTX sequences and reveal the presence of
an alternative CTX attachment site.
To begin to address whether CTX integration into the Bah-2
chromosome occurs at more than one chromosomal location, we performed Southern analyses on DNA isolated from five Bah-2Kn strains that had
been transduced to kanamycin resistance by the CP-T1ts lysate made on
SM115. We found two additional hybridization patterns with DNA
digested with BglII and probed with an rstA probe
(data not shown). Thus, in the absence of attRS,
CP-T1ts-transduced CTX-Kn can integrate at multiple sites on the
Bah-2 chromosome. Some Bah-2Kn transductants did not harbor integrated
CTX but only pCTX-Kn. The factors which determine whether or not
CTX integrates into the attRS background are unknown.
Analysis of Knr transductants of Bah-3, a mutant
recA derivative of Bah-2 (46), demonstrated the
requirement for homologous recombination in the integration of
CP-T1ts-transduced CTX DNA in a attRS background. No
evidence of chromosomal integration of CTX within Bah-3Kn was found
by Southern analysis (the weak band seen in Fig. 3 reflects background
hybridization). Instead, in Bah-3Kn the CTX was recovered only as a
plasmid copy (Fig. 2). Thus, unlike CTX integration into
attRS, which can occur independent of recA
(33), integration of CP-T1ts-transduced CTX into a
attRS background is recA dependent.
Relative efficiency of CP-T1ts ctxAB transduction.
Our preliminary observations suggested that CP-T1 preferentially
transduces ctxABN4::Kn, as indicated
by the high number of Knr transductants recovered for each
of the four V. cholerae recipient strains. To test the
relative efficiency of ctxABN4::Kn
CP-T1ts transduction, we first constructed an SM115 derivative,
SM115-Tc, which contains lacZ::Tc. We
then made a CP-T1ts lysate on SM115-Tc and compared the number of
Knr 2740-80 transductants (indicative of ctxAB
transduction) with the number of Tcr 2740-80 transductants
(indicative of lacZ transduction). The Kn-marked
ctxAB was transduced from SM115-Tc more than 200 times more
frequently than the Tc-marked lacZ into the same recipient cells. There were 980 Knr 2740-80 transductants and only 4 Tcr 2740-80 transductants. Similarly, when this lysate was
used to transduce Bah-2, we found 1,146 Knr Bah-2
transductants and only 5 Tcr Bah-2 transductants.
is the presence of pCTX-Kn in SM115. To
determine whether CP-T1ts can package pCTX-Kn DNA, we made use of the
Bah-3Kn transductants that we found only contain the plasmid form of
CTX-Kn. CP-T1ts was grown on this strain, and then this lysate was
used to transduce 468-83, 2740-80, and Bah-2. We obtained
Knr transduced cells for all three strains, indicating that
CP-T1ts can package pCTX-Kn DNA efficiently.
Host range of CP-T1ts
ctxABN4::Kn transduction.
To
examine whether CP-T1 could transduce ctxAB to non-O1
V. cholerae strains, we attempted to transduce strains of 20 different non-O1 serogroups of V. cholerae by using the
same CP-T1ts lysate made on SM115 that had been used to
transduce the El Tor O1 strains described above. Interestingly, only
one non-O1 serogroup was transduced to Kn resistance by CP-T1ts,
serogroup O37. As previously reported (20), this finding
suggests that the CP-T1ts receptor is the O1 serogroup antigen and that
infection of non-O1 strains by this phage is very unusual.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacterial species exchange DNA by the parasexual means of conjugation, transduction, and transformation. These mechanisms of horizontal gene transfer play an important role in increasing the genetic variability of a bacterial species and also confer new phenotypes, such as virulence, to the recipient. Horizontal transfer of the genes encoding O139 lipopolysaccharide to an El Tor V. cholerae strain is thought to underlie the emergence of V. cholerae O139, a novel epidemic variant of V. cholerae (3-5, 12, 41, 44). This remarkable event stimulated renewed interest in the role of horizontal gene transfer in the emergence and evolution of pathogenic V. cholerae strains.
More recently, it has been demonstrated that CT is encoded by a
lysogenic inovirus, CTX (45). Interestingly, the receptor for CTX, TCP (42), is itself encoded by a recently
described novel lysogenic inovirus, VPI (22-24). Hence,
it has been presumed that only nontoxigenic V. cholerae
strains that already contain TCP can acquire ctxAB via
lysogenic conversion by CTX infection. However, the exact molecular
sequence of the acquisition of CTX is unsettled, with reports in the
literature of toxigenic strains of V. cholerae that lack the
genes encoding TCP (18, 38). Some authors have suggested
that such isolates arose by TCP-mediated CTX infection, with
subsequent loss of the TCP island (VPI) (14, 15, 22).
Alternatively, such strains may possess an as-yet-unidentified receptor
and/or modes of CTX acquisition that account for the presence of a
CTX prophage in these strains.
In our current study, we show that the generalized transducing phage
CP-T1 can transfer the entire CTX genome to a number of V. cholerae strains. We found that the CP-T1-transduced CTX integrates into strain 468-83, a V. cholerae strain that
lacks both the CTX receptor TCP and the attRS attachment
site. Therefore, it appears that TCP may not always be a limiting
factor in the conversion of nontoxigenic strains to toxigenicity. Also,
these results suggest that disruption of gene products required for TCP-mediated CTX uptake in live-attenuated vaccine strains will not
completely protect vaccine strains from reacquiring ctxAB.
Integration of CTX into the chromosome of isolates lacking
attRS (e.g., 468-83 and Bah-2) indicates that there are
alternative attachment sites or mechanisms of integration in these
strains for CTX. Examination of multiple independent CP-T1ts
transductions of CTX into strain Bah-2 also showed the presence of
at least three alternative CTX attachment sites in this strain and
demonstrates that the attRS sequence is nonessential for
CTX integration. Among the four El Tor V. cholerae
strains we used as recipients to study CP-T1 transduction of CTX,
three different patterns of CTX integration into the chromosome were
found. Strain 468-83 lacks homology to attRS, CTX, RS1,
and TLC sequences but contains the rtxA gene. A
Knr transductant of this strain gave a hybridization
pattern almost identical to the donor strain SM115, suggesting that
CTX and flanking DNA integrated into the 468-83 chromosome via
homologous recombination involving the 5' end of the RTX region. In
contrast, a CP-T1ts transductant of strain 2740-80, which contains the
attRS attachment site and both the TLC and the RTX regions,
contains a single copy of CTX flanked by RS1 sequences integrated
into the chromosome. Integration in this strain probably occurred by CTX-mediated site-specific recombination. The
chromosomal arrangement of the 2740-80Kn transductant suggests
that RS1-CTX-RS1 may integrate as a single genetic element. Bah-2Kn
transductants contained two CTX prophages in tandem, and no RS1, TLC,
or RTX sequences were transduced. The mechanism by which this strain
obtained two tandem copies of CTX that lacked an RS1 sequence is not clear.
The morbidity and mortality of cholera, as well as the significant
negative effects of this dreaded diarrheal disease on the economics of
developing countries, make this disease a major public health problem
and a target for the development of a safe and effective vaccine.
Ideally, to prevent vaccine reversion to toxigenicity, live-attenuated
V. cholerae vaccine strains should be resistant to CTX
infection. Mekalanos and coworkers have engineered El Tor-derived live
vaccine strains which include a deletion of the CTX attachment site,
attRS, in pursuit of this goal (33). We report
here that CP-T1ts CTX-Kn transductants of the vaccine strain Bah-2
lacking attRS still contained chromosomally integrated CTX-Kn, indicating that even this deletion of the CTX attachment site does not entirely safeguard this type of vaccine construct against
lysogenic conversion. However, CP-T1ts CTX-Kn transductants of
Bah-3, a recA derivative of Bah-2, only contained the
plasmid form of CTX-Kn, thereby demonstrating the necessity for
recA in the chromosomal integration of CTX into
attRS strains, as well as the value of this deletion in
live vaccine constructs.
Since our examination of the host range of CP-T1 transduction of CTX
revealed that only 2 of 20 different V. cholerae serogroups, i.e., O1 and O37, were transduced, CP-T1 may not be important in the
transfer of ctxAB to most non-O1 strains. Interestingly, the
O37 strain has been shown to be closely related to epidemic O1 V. cholerae strains and was associated with a serious cholera outbreak in Sudan in 1968 (2, 5, 49). Although the CP-T1 host range appears to be limited to a subset of V. cholerae
serogroups, it has been proposed that non-O1 serogroups may convert to
the O1 serogroup and vice versa under certain environmental conditions, which may allow for the extension of the CP-T1 V. cholerae
host range (10, 11). Furthermore, the importance of CP-T1 in
particular and transducing phage in general cannot be overemphasized.
Since the aquatic ecosystems that constitute the natural habitat of V. cholerae are known to contain enormous numbers and
varieties of bacteriophages (13, 34, 48), it is reasonable
to hypothesize that other V. cholerae generalized
transducing phages exist, which transfer genes among V. cholerae populations independent of the serogroup. Our studies not
only demonstrate an alternative evolutionary scenario for the emergence
of toxigenic V. cholerae isolates but also suggest the
potential, under the right selective pressures, for the horizontal
transfer of CT genes to a broader range of bacterial isolates.
We found that ctxAB is preferentially transduced by CP-T1.
Comparison of the frequency of
ctxABN4::Kn transduction with the frequency of lacZ::Tc transduction from
the same strain demonstrated that CP-T1 transduction of CTX was
markedly more efficient than the transduction of
lacZ::Tc. The reason that CP-T1
preferentially transduces CTX DNA is unknown, but several
possibilities can be envisioned. Previously, others have identified
potential CP-T1 pac sites (20); we therefore searched the
DNA sequence of CTX and its flanking regions for CP-T1 pac sites and
identified a large number of candidates, which might explain the
relatively high frequency of
ctxABN4::Kn transfer compared with
lacZ::Tc transfer. Alternatively, and
not mutually exclusive with this possibility, the preferential
transduction of CTX DNA may be in part accounted for by the higher
copy number of CTX sequences in SM115, the donor strain used in
these transductions. The presence of both the tandemly duplicated
CTX-Kn prophage and the CTX-Kn RF in SM115 increase the copy number
of CTX-Kn DNA in this strain. It is also possible that there is
preferential packaging by CP-T1 of the CTX-Kn RF (which is present
in SM115) compared to chromosomal sequences. In fact, we found that
CP-T1 could efficiently transduce pCTX-Kn from Bah-3, a strain that
lacks chromosomal CTX sequences. Since CP-T1 was initially isolated
from V. cholerae (21, 32), it may constitute an
important mechanism of horizontal ctxAB DNA transfer in
V. cholerae populations. Furthermore, the elevated frequency
of CTX transduction by CP-T1 may suggest coevolution of these bacteriophages.
The preferential transduction of CTX by phage CP-T1 illustrates the
potential importance of interactions between bacteriophages in the
horizontal transfer of genes encoding virulence factors. The roles that
interactions between bacteriophages play in the evolution of bacterial
pathogens is becoming increasingly recognized. For example, Lindsay et
al. recently described a phage-like element that carries the gene for
toxic shock syndrome toxin 1 (TSST-1), which is excised and
circularized by staphylococcal phages 
13 and 80
(28).
Moreover, 80 transduces TSST-1 at high frequency and may be
responsible for the spread of TSST-1 production among Staphylococcus aureus strains (28).
Figueroa-Bossi and Bossi recently described how interactions between
the Gifsy-1 and Gifsy-2 prophages play a role in
Salmonella enterica serovar Typhimurium virulence
(17). Furthermore, recent studies have demonstrated that
R-type pyocin particles induced from Pseudomonas aeruginosa C contain closed circular single-stranded DNA that shows homology to
filamentous phages (26). Finally, the demonstration that TCP, an essential V. cholerae colonization factor and the
receptor for CTX, is itself encoded by a filamentous phage (VPI)
indicates that a cell-surface form of a phage (TCP) can act as a
receptor for another phage, a novel bacteriophage interaction. Whether bacteriophage interactions generally occur primarily within the human
host, as seems to be the case for VPI and CTX (25, 29, 45), or within other environments remains to be investigated. In
either case, the complexity of the apparent coevolution of diverse
bacteriophages and their bacterial hosts in the evolution of bacterial
pathogens is remarkable.
| |
ACKNOWLEDGMENTS |
|---|
We thank A. Camilli and D. Hava for kindly providing us with the CP-T1ts bacteriophage and for useful suggestions. We thank our colleagues, Andrew Heilpern, Katie Moyer, Brigid Davis, and Bianca Hochhut, for critically reading the manuscript. We are also grateful to Anne Kane and the New England Medical Center GRASP Center (grant P30DK-34928) for providing us with culture media.
This work was supported by National Institutes of Health grant AI-42347 to M.K.W. E.F.B. was supported by training grant T32 AI-07329. M.K.W. is a PEW Scholar of Biomedical Research.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Division of Geographic Medicine/Infectious Disease, Tufts-New England Medical Center, 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
| |
REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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