Microbiology (2004), 150, 1073–1078
DOI 10.1099/mic.0.26781-0
A DNA adenine methylase mutant of Shigella
flexneri shows no significant attenuation of
virulence
Yasuko Honma, Reinaldo E. Fernández and Anthony T. Maurelli
Correspondence
Anthony T. Maurelli
[email protected]
Received 23 September 2003
Revised 17 December 2003
Accepted 22 December 2003
Department of Microbiology and Immunology, F. Edward Hébert School of Medicine,
Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda,
MD 20814-4799, USA
Mutants of Salmonella defective in DNA adenine methylase (dam) have been reported to be
attenuated for virulence and to provide protective immunity when used as vaccine strains. To
determine whether these observations could be extended to Shigella, a dam mutant of Shigella
flexneri 2a was characterized and examined for the role of dam in pathogenesis. The Shigella
dam mutant showed some unique characteristics; however, it retained virulence in vivo as well
as in vitro. The mutant invaded cultured L2 monolayer cells as efficiently as the wild-type parent,
but its intracellular growth was suppressed up to 7 h post-invasion. Furthermore, the invading
dam mutant formed smaller plaques in cell monolayers compared to the parent strain. However,
the mutant produced keratoconjunctivitis in the Sereny test in guinea pigs only slightly more
slowly than the wild-type. While the effect of the dam mutation on virulence was modest, the
rate of spontaneous mutation in the dam mutant was 1000-fold greater compared with the
wild-type. The virulence and high mutability displayed by the dam mutant of Sh. flexneri
suggest that a general anti-bacterial pathogen vaccine strategy based on mutations in dam
needs to be re-evaluated.
INTRODUCTION
Shigella flexneri, one of the causative agents of bacillary
dysentery, invades and spreads in epithelial cells of the colon
and causes extensive destruction of the mucosal surface.
Virulence of Shigella spp. is multigenic, involving genes both
on the chromosome and on a large virulence-associated
plasmid (Parsot & Sansonetti, 1996; Sansonetti et al., 1982).
DNA methylation is a mechanism by which bacteria regulate
gene expression and control cellular functions such as DNA
replication, mismatch repair, and segregation of chromosomal DNA (Marinus, 1996). Enzymes known as DNAmethyltransferases mediate methylation, which occurs at the
C-5 or N-4 positions of cytosine and at the N-6 position of
adenine. DNA-methyltransferases are found in many genera
of enteric bacteria, but DNA methylation is not essential for
viability of Escherichia coli or Salmonella (Garcı́a-del Portillo
et al., 1999; Low et al., 2001).
Recently, DNA adenine methylase (Dam), which methylates the N-6 of adenine in GATC sequences, has been
reported to play a key role in regulation of virulence of
several bacterial pathogens. Dam2 Salmonella typhimurium
display reduced M-cell cytotoxicity and reduced invasion in
tissue culture (Garcı́a-del Portillo et al., 1999). In an animal
Abbreviation: Ipa, invasion plasmid antigen.
0002-6781
Printed in Great Britain
model, these mutants colonize Peyer’s patches but fail to
invade enterocytes and fail to cause disease (Heithoff et al.,
1999). It has also been reported that Dam is required for
expression of virulence genes in Yersinia pseudotuberculosis
and Vibrio cholerae. In these organisms, Dam activity is
essential for viability. However, overexpression of dam
inhibits colonization of V. cholerae in the suckling mouse
model and strongly attenuates virulence of Y. pseudotuberculosis in a murine model (Julio et al., 2001). Heithoff et al.
(1999, 2001) showed that dam mutants of Salmonella
provide effective immunity when used as live attenuated
vaccines in the mouse model. These authors also proposed
that because of the high conservation of DNA adenine
methylases among pathogenic bacteria, these proteins are
potential targets for the development of vaccines and antimicrobial agents.
In light of these reports, it has been suggested that Dam is
likely to be important for pathogenesis of other bacterial
pathogens as well as those just described (Low et al., 2001).
In this study, we tested this hypothesis by constructing a
dam mutant of Shigella flexneri 2a and examining the virulence properties of the mutant. We report that, contrary to
what has been observed in Salmonella, a dam mutant of Sh.
flexneri retains the ability to invade, grow intracellularly and
spread intercellularly, and is only minimally altered in its
virulence phenotypes.
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Y. Honma, R. E. Fernández and A. T. Maurelli
METHODS
after centrifugation. At the indicated intervals, cell monolayers (in
triplicate) were washed to remove extracellular bacteria and gentamicin, lysed with 0?5 % Triton X-100 in H2O to release intracellular
bacteria, and the recovered bacteria were plated on Congo red agar
for counting.
Bacterial strains, growth conditions and mutant construction.
2457T, a wild-type strain of Sh. flexneri 2a (Formal et al., 1958), and
BS103, a virulence plasmid-cured derivative of 2457T (Maurelli et al.,
1984), were used. dam mutants were obtained by P1L4 transduction
of the dam-13 : : Tn9 mutant allele from E. coli K-12 (Marinus et al.,
1983) into 2457T and BS103. Chloramphenicol-resistant transductants were selected and designated 2457T/dam and BS103/dam,
respectively. The dam null phenotype was confirmed by digestion of
plasmid DNA isolated from the dam mutants with MboI, which
cleaves only unmethylated GATC sequences. Bacteria were grown in
tryptic soy broth (TSB), Luria broth (LB) or on Congo red agar
(TSB, 0?02 % Congo red, 1?5 % Bacto agar) with or without chloramphenicol (10 mg ml21).
Spontaneous mutation rate. To determine the mutation rate in
dam mutants of Shigella, spontaneous mutation to rifampicin resistance was examined, as described previously (Bale et al., 1979).
Briefly, a few hundred cells from each strain were inoculated into
5 ml LB and the cells were cultured overnight to saturation at 37 uC
with shaking. Bacteria from each culture were plated on LB agar,
with or without 100 mg rifampicin ml21. After incubation overnight
at 37 uC, the plates were scored for rifampicin-resistant colonies and
the frequency of spontaneous mutation calculated.
Statistical analysis. Values are reported as mean±standard error of the
mean (SEM) in tables. All results were analysed using Student’s t-test.
Secretion of invasion plasmid antigens (Ipas). Secretion of IpaB
and IpaC from 2457T and 2457T/dam was examined as previously
described (Bahrani et al., 1997).
Virulence assays. Invasion assays with 2457T and 2457T/dam were
RESULTS
performed using a gentamicin protection assay with semiconfluent
monolayers of L2 mouse fibroblasts, as described before (Hromockyj
& Maurelli, 1989). Plaque assays were performed using confluent L2
monolayers, as described by Oaks et al., (1985). The Sereny test was
used as an in vivo virulence assay (Hartman et al., 1991).
Transduction of dam-13 : : Tn9 into 2457T and
BS103
dam mutants were obtained by transducing the dam13 : : Tn9 mutant allele from E. coli K-12 into Sh. flexneri
2457T and BS103. Chloramphenicol-resistant transductants of 2457T (2457T/dam) and BS103 (BS103/dam) were
chosen and analysed further. The dam null phenotype was
confirmed by digestion of plasmid DNA isolated from the
mutants with restriction endonuclease MboI, which cleaves
only unmethylated DNA sequences. Plasmid DNA isolated
from dam mutants was sensitive to cleavage by MboI, while
plasmid DNA isolated from the isogenic parent strains was
completely resistant (data not shown).
Bacterial growth in liquid culture. Strains 2457T, 2457T/dam,
BS103 and BS103/dam were cultured overnight with shaking at 30 uC,
subcultured 1 : 100 into fresh TSB, and then incubated at 30 uC or
37 uC (for 2457T and 2457T/dam), or at 37 uC (for BS103 and BS103/
dam). Samples were taken every hour and plated to determine viable
cell numbers.
Measurements of intracellular bacterial growth. Assays for
growth of intracellular bacteria were carried out as previously
described, with some modifications (Schuch et al., 1999). Briefly,
strain 2457T and 2457T/dam were grown to early–mid log phase in
TSB. Bacteria were washed with PBS and suspended in Dulbecco’s
minimal essential medium (DMEM) to an OD600 of 0?72. One
millilitre of this bacterial suspension (containing about 6–86
108 c.f.u.) was applied to each monolayer of semiconfluent L2 cells
in 35 mm tissue culture dishes. After 10 min centrifugation at
1000 g and a 30 min invasion period at 37 uC in CO2, infected
monolayers were washed with PBS and further incubated in DMEM
containing 50 mg gentamicin ml21. For measurement of intracellular
growth, t=0 was set at the beginning of the incubation period
Secretion of Ipa proteins
The mutant strain 2457T/dam was first tested for the ability
to secrete invasion effectors via the Shigella type III secretion
system. This phenotype is tightly regulated by growth temperature and is essential for invasion and intracellular growth.
2457T/dam synthesized as much IpaBC as the parent 2457T,
Table 1. Virulence assays of strain Sh. flexneri 2a 2457T and its dam mutant derivative
Values are reported as mean±SEM.
Strain
Invasion ratio*
Plaque-forming
ratioD
Plaque size after
3 days (mm)
Plaque size after
5 days (mm)
Sereny test
(no. positive/no. tested)
2457T
2457T/dam
0?0022±0?0004
0?0017±0?0003d
0?0612±0?0088
0?0309±0?0103§
2?0–2?5
1?0–2?1
3?6–4?0
1?7–3?5
2/2||
3/3
*Expressed as number of gentamicin-protected c.f.u. per input c.f.u.
DExpressed as number of plaques per input c.f.u.
dP value in t-test was 0?1 compared to the wild-type strain.
§P value in t-test was 0?04 compared to the wild-type strain.
||Positive reaction 48 h after inoculation.
Positive reaction 48–96 h after inoculation.
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Microbiology 150
dam and Shigella pathogenesis
c.f.u. per well
108
2457T
2457T/dam
107
106
105
104
1
2
3
4
5
Time (h)
6
7
8
Fig. 3. Intracellular growth of Sh. flexneri 2a 2457T and
2457T/dam in L2 cells. Beginning of the incubation was
defined as t=0. Experiments were repeated three times and
one representative experiment is shown. Each data point represents the mean±SD of triplicate data in the experiment.
Fig. 1. Plaque formation in an L2 monolayer by a dam mutant
of Sh. flexneri 2a. A, wild-type parent 2457T; B, isogenic dam
mutant 2457T/dam. Plaque formation is shown 3 days postinvasion. Culture dish diameter, 35 mm.
Bacterial growth in liquid culture and in
invaded cells
Because of the observed difference in plaque size, we measured in vitro and in vivo growth of the dam mutants. The
strains were cultured in TSB at 30 uC or 37 uC (for 2457T and
2457T/dam), or at 37 uC (for BS103 and BS103/dam). The
generation time in exponential phase at 37 uC was about
30 min for 2457T and 45 min for 2457T/dam (Fig. 2). The
generation time at 30 uC of 2457T/dam was essentially
unchanged from 2457T: 41 min and 39 min, respectively
(Fig. 2). The generation times of both BS103 and BS103/
dam at 37 uC were identical, at about 30 min (Fig. 2).
and secreted similar amounts of these effector proteins into
the growth medium, as detected by Western blotting (data
not shown).
Virulence assays
Next, 2457T/dam was tested for the ability to invade tissue
culture cells and to spread from cell to cell (as measured in
the plaque assay). No significant difference in the efficiency
of invasion was detected between 2457T and 2457T/dam
(Table 1, P=0?1) and only a modest difference in plaque
formation ratio was found (Table 1, P=0?04). Plaques
formed by 2457T/dam tended to be smaller in size compared to 2457T (Fig. 1). In the Sereny test, 2457T/dam produced keratoconjunctivitis in all three of the animals tested,
although fully developed keratoconjunctivitis was delayed
in two of the animals (Table 1).
1010
2457T
2457T/dam
108
1
2
3 4 5
Time (h)
6
7
_1
1010
_1
1011 37 ˚C
c.f.u. ml
109
107
1011 30 ˚C
109
108
2457T
2457T/dam
107
8
106
c.f.u. ml
37 ˚C
1010
c.f.u. ml
_1
1011
Assays for growth of intracellular bacteria were carried out,
and the slower growth rate of 2457T/dam was again observed (Fig. 3). At 2 h post-invasion, the number of recoverable c.f.u. of 2457T began to increase, and bacterial numbers
continued to increase until 4 h post invasion. At this point,
c.f.u. recovered began to decrease as infected L2 cells died
and detached from the plates. 2457T/dam displayed no
1
2
3
4 5
Time (h)
6
7
8
109
108
BS103
107
BS103/dam
106
1
2
3
4 5
Time (h)
6
7
8
Fig. 2. Growth curves of Sh. flexneri 2a 2457T, BS103, and their isogenic dam derivatives. Strains were grown in TSB at
30 6C or 37 6C. Samples were taken every hour and plated to determine viable bacteria. Each data point represents the
mean±SD of three independent experiments.
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Y. Honma, R. E. Fernández and A. T. Maurelli
intracellular growth up to 7 h, and only then started to show
an increase in bacterial numbers (Fig. 3). Cells infected by
2457T/dam were still attached to the culture plates at 8 h
post-invasion (data not shown). Therefore, it is unlikely that
the decrease in viable counts of the dam mutant was due to
detachment of dam-infected cells from the monolayer.
Morphology of intracellular bacteria
Giemsa staining of infected monolayers was performed to
visualize intracellular bacteria. The wild-type parent 2457T
multiplied rapidly within the infected cells, filling the cytoplasm within 4 h (Fig. 4). The infected cells also displayed
numerous cytoplasmic extensions that contained bacteria
(data not shown), a phenotype that is associated with intracellular motility. After 7 h, most cells had detached from the
culture plate, but the cells that remained attached contained
many bacteria. In contrast, 2457T/dam formed long, snaking filaments in the L2 cells after invasion, in addition to
normal-length bacteria (Fig. 4). About 20 % of the infected
cells contained long filamentous bacteria at each time point
observed post-infection. There were no cells that were filled
only with long filamentous bacteria.
Spontaneous mutability
The effect of the dam mutation on the rate of spontaneous
mutation was measured by determining the frequency of
spontaneous resistance to rifampicin. 2457T/dam showed a
mutation frequency of 2?2±0?761026, which was about
1000-fold higher than that of the parent, 2457T (2?3±0?76
1029). For comparison, the spontaneous mutation rate in
E. coli dam mutants is only 20- to 80-fold higher than the
wild-type (Bale et al., 1979; Glickman, 1979).
DISCUSSION
Many different genetic approaches have been used to attenuate pathogenic strains of bacteria in order to produce live
vaccine strains capable of generating protective immunity
without causing disease. It has recently been reported
that DNA adenine methylase is required for expression of
virulence genes in Salmonella enterica (Heithoff et al., 1999),
Yersinia pseudotuberculosis and Vibrio cholerae (Julio et al.,
2001). A dam mutant of Salmonella typhimurium shows
reduced invasion of enterocytes, and is avirulent when given
orally and interperitoneally (Garcı́a-del Portillo et al., 1999).
Moreover, Dam2 Salmonella, administered orally or intraperitoneally, provides significant protection against challenge
with wild-type Salmonella (Heithoff et al., 1999). Accordingly, although the mechanism of the dam requirement for
Salmonella virulence remains unknown, it has been proposed that dam mutants could serve as live attenuated
vaccines and that Dam itself may provide a potential target
for broad antimicrobial activity (Heithoff et al., 1999).
In this study, we constructed a dam mutant of Sh. flexneri 2a
strain 2457T. The mutant 2457T/dam was slightly attenuated in virulence in tissue culture invasion assays, plaque
assays and, in vivo, in the guinea pig Sereny test. However,
2457T/dam formed smaller plaques in the plaque assay, and
gave a delayed positive reaction in some of the animals in
the Sereny test, compared to 2457T. Growth in TSB was
also slower for the dam mutant, and in invaded cells some
2457T/dam bacteria were filamentous. Genes involved in
stress and the SOS response are expressed at higher levels in
dam mutants of E. coli (Marinus, 1996; Oshima et al., 2002),
and the filamentous shape of some dam mutant Sh. flexneri
in invaded cells suggested an increased SOS response in the
Sh. flexneri dam mutant.
Fig. 4. Morphology of wild-type Sh. flexneri 2a 2457T and its dam mutant derivative inside infected L2 fibroblasts. Panels
show monolayers at different time points, as defined in Fig. 3. Bar at 1 h and 4 h, 10 mm; bar at 7 h, 20 mm.
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Microbiology 150
dam and Shigella pathogenesis
We were rather surprised that the efficiency of invasion of
the Sh. flexneri dam mutant was indistinguishable from that
of the wild-type parent, in spite of its high mutability and
slower growth. In contrast, a Sal. typhimurium dam mutant
is non-invasive in tissue culture (Garcı́a-del Portillo et al.,
1999). The Sh. flexneri dam mutant was also virulent in
an animal model, while a Sal. typhimurium dam mutant is
completely attenuated in an animal model (Heithoff et al.,
2001). It has been proposed that since motility is decreased
in a dam mutant of E. coli (Oshima et al., 2002), this phenotype might be responsible for the lack of invasiveness by the
mutant of Sal. typhimurium. Motility is important for invasiveness of Salmonella typhi (Liu et al., 1988), and increases
the rate of invasion of Sal. typhimurium (Khoramian-Falsafi
et al., 1990; Moens & Vanderleyden 1996), but it is not
important for invasion of Shigella, which is non-motile. The
different behaviour of the dam mutants underscores the fact
that the fundamental mechanism of pathogenesis of Shigella
differs from that of Salmonella.
DNA adenine methylase in bacteria regulates a variety of
functions, including chromosome replication, transcription, and DNA repair (Marinus, 1996). Spontaneous mismatch mutations that occur in rapidly growing bacteria are
normally repaired by the dam-directed mismatch repair
system. Thus, one phenotype of dam mutants is that they
display an increased rate of mutation. We found that 2457T/
dam had a mutation rate about 1000-fold higher compared
to the wild-type parent. This result is in stark contrast with
the spontaneous mutation rate of a dam mutant of E. coli,
which was reported to be only 20- to 80-fold higher than
wild-type (Bale et al., 1979). The molecular basis for this
increased mutation rate remains to be elucidated.
These results suggest that a general anti-bacterial pathogen
vaccine strategy based on dam mutants needs to be reevaluated. Although inactivation or overexpression of dam
has a strong attenuating effect on pathogens such as Salmonella, Yersinia and V. cholerae (Heithoff et al., 2001; Julio
et al., 2001), a mutation in dam does not significantly alter
virulence in Sh. flexneri. Although the Sh. flexneri dam
mutant produced a positive reaction in guinea pigs in the
Sereny test, we cannot exclude the possibility that the
mutant might be attenuated in a human host. However,
the increased rate of spontaneous mutation we observed in
the Sh. flexneri dam mutant would not be a desirable trait for
a live vaccine strain.
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ACKNOWLEDGEMENTS
Mol Gen Genet 192, 288–289.
We thank Dr Marjan van der Woude for providing the E. coli strain
carrying the dam-13 : : Tn9 allele and for helpful discussions for this
manuscript. This work was supported by grant AI24656 from the
National Institute of Allergy and Infectious Diseases.
The opinions or assertions contained herein are the private ones of the
authors and are not to be construed as official or reflecting the views of
the Department of Defence or the Uniformed Services University of the
Health Sciences.
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