Dam and its role in pathogenicity of Salmonella enterica.

Abstract

Dam methylation is an essential factor involved in the virulence of an increasing number of bacterial pathogens including Salmonella enterica. Lack of Dam methylation causes severe attenuation in animal models. It has been proposed that dysregulation of Dam activity is potentially a general strategy for the generation of vaccines against bacterial pathogens. In this review, we focus our attention on the role of methylation by Dam protein in regulating bacterial gene expression and virulence in Salmonella enterica.

Full text

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Review Article

Dam and its role in pathogenicity of Salmonella enterica

Mónica N. Giacomodonato, Sebastián H. Sarnacki, Mariángeles Noto Llana, and M. Cristina
Cerquetti.

Centro de Estudios Farmacológicos y Botánicos CEFyBO-CONICET, Departamento de Microbiología, Parasitología e
Inmunología, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina

Abstract
Dam methylation is an essential factor involved in the virulence of an increasing number of bacterial pathogens including Salmonella
enterica. Lack of Dam methylation causes severe attenuation in animal models. It has been proposed that dysregulation of Dam activity is
potentially a general strategy for the generation of vaccines against bacterial pathogens. In this review, we focus our attention on the role of
methylation by Dam protein in regulating bacterial gene expression and virulence in Salmonella enterica.

Keywords: DNA adenine methylase, Pathogenesis, Salmonella enterica

J Infect Dev Ctries 2009; 3(7):484-490.

Received 10 March 2009 - Accepted 4 July 2009

Copyright © 2009 Giacomodonato et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction
DNA methylation takes place throughout the
living world, including in bacteria, plants, and
mammals. DNA methylation occurs at the C-5 or N-
4 positions of cytosine and at the N-6 position of
adenine and is catalyzed by enzymes known as DNA
methyltransferases (MTases) [1,2]. All MTases use
S-adenosyl methionine as a methyl donor.
DNA methylation has historically been
associated with DNA restriction modification
systems. These systems are considered to be
important in protecting cells from foreign DNAs such
as transposons and bacteriophages [3,4,5].
Restriction modification systems contain a DNA
methylase that protects host DNA sequences from
restriction with their cognate restriction enzymes,
which digest unmodified foreign DNAs. Certain
MTases, including DNA cytosine MTase (Dcm),
which methylates the C-5 position of cytosine in
CC(A/T)GG sequences; DNA adenine methylase
(Dam), which methylates N-6 of adenine in GATC
sequences; and cell cycle regulated methylase
(CcrM), which methylates the N-6 adenine of
GAnTC, do not have cognate restriction enzymes
associated with them [6].
Methylation of DNA has numerous consequences
for bacterial physiology, including the regulation of
chromosome replication, DNA segregation,
mismatch repair, transposition, and transcriptional
regulation. The molecular basis for the pleiotropic
phenotypes associated with Dam is the differential
methylation of DNA resulting in an altered affinity of
regulatory DNA binding proteins. Regulatory
proteins might preferentially bind to non-methylated
DNA, thus blocking methylation by Dam, while other
proteins bind with high
hemimethylated or fully methylated DNA [7].
Dam homologues are widespread among enteric
bacteria, including Escherichia coli, Salmonella spp.,
Serratia marcescens, Yersinia spp., and Vibrio
cholerae, but are also present in disparate genera,
including Neisseria, among others. Dam is a vital
protein in
Vibrio
cholerae
pseudotuberculosis [8,9]; however, Dam methylation
is not essential for the viability of E. coli or
Salmonella spp. [10]. On the other hand, methylation
by Dam is fundamental in Salmonella virulence, and
its absence causes severe attenuation in animals
(mice, chickens, cattle) [11,14]. Following oral
immunization with dam Salmonella, mice infected
with 10,000 times the LD50 of the wild type strain
were highly protected and exhibited reduced
proliferation in systemic tissues compared with the
wild type strain. However, dam mutants are able to
persist in the liver, spleen, and lymph nodes of
infected mice [11,12].
Moreover, dam mutants of Salmonella enterica
serovar Typhimurium exhibit virulence related
affinity only to
and
Yersinia

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defects such as impaired expression and secretion of
invasion proteins [11], reduced cytotoxicity on M
cells [11], inefficient colonization of Peyer’s patches
and mesenteric lymph nodes [11,12], sensitivity to
components of innate immunity [15,16,17], envelope
instability [18]; changes in LPS expression [Sarnacki
et al., unpublished data] and probably additional
defects still to be discovered since the pleiotropic
effects of Dam methylation. In this review, we focus
our attention on the role of Dam methylation in
regulating bacterial gene expression and virulence in
S. enterica.

Dam and the regulation of LPS synthesis
The lipopolysaccharide
component of the outer membrane of gram-negative
bacteria that contributes to the stability and
permeability-barrier properties of this membrane.
LPS is located on the outer leaflet of the outer
membrane and consists of three regions: O antigen
polysaccharide, core oligosaccharide, and lipid A
[19]. The biogenesis of LPS is a complex process
involving various steps that occur at the plasma
membrane followed by the translocation of LPS
molecules to the outer membrane [19,20]. On the
periplasmic side of the
translocated subunits polymerize to a certain length,
unique to each O antigen, by the concerted functions
of Wzy (O-antigen polymerase) and Wzz (O-antigen
chain length regulator). The polysaccharide is
ultimately ligated to the lipid A-core oligosaccharide.
We reported earlier that a Salmonella enterica
serovar Enteritidis mutant expressing a truncated
Dam protein [17,21] fails to agglutinate with anti O9
serum [22]. More recent studies demonstrated that
the Dam protein of S. Enteritidis has an effect on the
determination of O antigen polysaccharide chain
length [Sarnacki et al., unpublished data] (Figure 1).
We found that Wzz protein is three-fold lower in the
Salmonella dam mutant than in the parental strain;
moreover, wzz gene expression is downregulated in
the mutant suggesting that a functional Dam is
required for adequate levels of wzz transcription.
Distinct two-component
PmrA/PmrB and RcsC/YojN/RcsB, are known to
regulate wzz gene expression independently of each
other [23]. Therefore, it is plausible that Dam could
regulate wzz gene expression either directly or by
affecting the expression of pmrA and rcsB genes.
Thus, our work uncovers rcsB and wzz as new targets
regulated by Dam methylation and demonstrates that
Dam has an effect on the determination of O antigen
(LPS) is a key
plasma membrane,
regulatory systems,
polysaccharide chain length [Sarnacki et al,.
unpublished data]. This phenomenon is not unique to
Salmonella as we also found that the lack of Dam
affects the LPS pattern in E. coli, increasing the
amount of shorter polysaccharides and modifying the
size of the banding pattern of the O antigen region
[Sarnacki et al., unpublished data]. Furthermore,
overproduction of Dam in Yersinia enterocolitica
results in an increased number of rough LPS
molecules [24]. These findings suggest that Dam
methylation plays a general role in the O antigen LPS
expression in enteric bacteria and could affect not
only the expression of bacterial surface proteins but
also the expression of surface polysaccharides.

Dam and the regulation of fimbrial genes
The S. Typhimurium genome contains a large
repertoire of putative fimbrial operons, including the
std operon, that remains poorly characterized because
these operons are not expressed under laboratory
conditions [25,26]. On the contrary, Std fimbriae are
synthesized during infection and play different roles
in virulence. Humphries et al. [26,27] observed that
mice infected with S. Typhimurium seroconvert to
StdA, the major protein of Std fimbriae detected upon
infection of bovine ileal loops, which provides
indirect evidence for in vivo expression of the std
operon. Deletion of std reduces intestinal persistence
of S. Typhimurium in the murine model. These data
suggest that Std fimbriae contribute to intestinal
colonization by mediating attachment to the intestinal
mucosa [28]. However, the underlying mechanisms
have not been explored. Dam methylation has
previously been implicated in controlling the
expression of fimbriae in E. coli [29,30] and S.
Typhimurium [31]. Balbontin et al. [32]
demonstrated that transcription of the std operon is
repressed by Dam methylation in S. Typhimurium.
They observed that stdA and stdB genes showed the
highest levels of Dam dependent regulation among
other genes. dam mutants of S. Typhimurium grown
under laboratory conditions express the std fimbrial
operon, which is tightly repressed in the wild type
strain [33]. Chessa et al. [34] confirmed and
extended this observation by showing that mutations
in dam resulted in the expression of StdA filaments
on the bacterial cell surface. The synthesis of Std
fimbriae has been shown to be silenced by S.
Typhimurium during static growth in LB medium,
whereas it is up regulated upon infection of bovine
ligated loops [26].

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Jakomin et al. [35] suggest that Dam methylation
regulates std expression at the transcriptional level.
Jakomin et al. [35] and Chessa et al. [34]
demonstrated that SeqA (GATC binding protein) and
RosE are repressors of the std operon and that HdfR
protein (a member of the LysR family of
transcriptional regulators) [36] is an activator of std
expression whose activity could be antagonized by
Dam methylation and SeqA. However, the
mechanisms underlying SeqA, RosE, and HdfR
mediated regulation of the std operon remain to be
investigated.
Overexpression of Std fimbriae and release to the
extracellular medium contributes to attenuation in
Salmonella dam mutants by activating the host
immune system. It has been suggested that the
ectopic fimbrial expression in dam mutants may
interfere with signal exchange between the host and
the pathogen and may overstimulate the host immune
system [37].

Dam and bacterial structure stability
Pucciarelli et al. [18] demonstrated that the
deficiency of Dam methylase causes envelope defects
in S. enterica [18]. This alteration was denoted by
the accumulation of membrane vesicles in the
supernatant of stationary cultures of dam mutants
[18]. The release of membrane material to the
extracellular medium correlated with a high protein
content in extracts prepared from culture supernatants
of dam mutants. The lack of Dam methylation in S.
enterica results in impaired association of membrane
proteins to peptidoglycan required for maintenance of
envelope integrity, such
peptidoglycan-associated
Braun’s murein lipoprotein (lppB). Surprisingly, E.

as OmpA, TolB,
and lipoprotein (Pal),

coli dam mutants do not show an equivalent
phenotype of envelope instability [18].
These observations suggest that Dam methylation
participate directly or indirectly in maintenance of
Salmonella envelope integrity. In this way,
Balbontín et al. [32] demonstrated a decreased
synthesis of the lppB gene in Salmonella dam
mutants. This LppB protein is involved in
maintaining envelope integrity [18]. Release of
membrane vesicles and leakage of proteins may
contribute to the virulence attenuation of S. enterica
dam mutants.
Another relevant defect of Salmonella dam
mutants that is linked to envelope instability is an
increased sensitivity to components of innate
immunity including antimicrobial peptides (defensins
NP-1 and bactinecin) [15], detergents (bile salts,
deoxycholate) [15,17,18,21], and mediators of
oxidative damage (H2O2) [15,21]. Envelope
instability may also lead to an increased release of
bacterial antigens.
In summary, the envelope defects, the enhanced
sensitivity to innate immune molecules together with
massive fimbrial expression on the cell surface
discussed above, may contribute to the attenuation of
virulence in S. enterica dam mutants in animals.
Enhanced release of membrane vesicles through
infected cells and tissues might cause overstimulation
of the host immune response. This ability of S.
enterica dam mutants to induce strong immune
responses in infected animals has been effectively
applied to the design of vaccines [11,12,15].

Dam and the secretion of effector proteins
S. enterica employ a type III secretion system (TTSS)
to translocate virulence determinants, called effector
proteins, from the bacterial cytoplasm into
Figure 1. Scheme of the outer membrane of wild type (wt) and dam Salmonella strains. The lack of Dam affects the LPS
pattern increasing the amount of shorter polysaccharides.

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the host cell cytoplasm thus increasing their
pathogenic potential and the outcome of infection.
TTSS involves the coordinated expression of
approximately 20 proteins and is therefore regulated
by complex mechanisms.
Effects of Dam on TTSS have been observed in
S. Typhimurium, although the basic regulatory
mechanisms have not been elucidated. Several
authors observed that both bacteria-associated and
secreted proteins are affected by the loss of Dam
regulation [11,15,18,38]. A report by Balbontín et al.
[32] provided evidence that Dam methylation
regulates the invasion genes of the pathogenicity
island 1 (SPI-1); they proposed a correlation between
specific alterations of gene expression and certain
virulence defects of Salmonella dam mutants. The
need for Dam methylation to activate the expression
of invasion genes could explain the reduced secretion
of SPI-1 effectors such as SipA, SipB and SipC
reported earlier [11] and the impaired invasion of
epithelial cells [11,38].
Recently, in our laboratory, we demonstrated that
Dam methylation regulates synthesis and secretion of
the Salmonella virulence effector SopA [38]. This
finding extends to an effector located outside SPI-1
(and secreted via the SPI-1 TTSS) described in
previous reports, indicating that Dam methylation
regulates Salmonella SPI-1. We observed a
significant reduction of SopA synthesis and secretion
under SPI-1 conditions in dam mutants of S.
Typhimurium. Interestingly, dam mutants expressed
SopA under noninducing conditions (28ºC), whereas




no synthesis of the effector was observed in the wild
type or the complemented strain at this temperature.
These results suggest that lack of Dam relaxes the
temperature regulation of SopA synthesis. Similar
results were observed in vitro by SipA, SopB, SopD,
and SopE2 effectors of S. Typhimurium dam strains
[46]. These observations are in agreement with
previous data obtained from Yersinia spp., showing
that Dam overproduction leads to the expression and
secretion of Yop virulence
nonpermissive conditions [8,40]. These authors
demonstrated that Dam overproduction disrupts both
thermal and calcium regulation of YopE synthesis
and relaxes the thermal (but not calcium) dependence
of YopE secretion. Moreover, we observed in vivo
that intracellular Salmonella dam mutants synthesize
SopA at lower levels than the wild type strain [38].
In summary, lack of Dam methylation causes an
atypical expression of many genes, and these changes
have been shown to attenuate the virulence of several
pathogens, including Salmonella spp., and to confer
protective immune responses in vaccinated animals
[7]. Dysregulation of Dam activity could lead to
ectopic gene expression and the concomitant
elaboration of an expanded repertoire of antigens. In
addition, the persistence of dam mutant vaccines of
Salmonella spp. in lymphoid tissues, such us Peyer’s
patches [11,15], may provide a stable source of
antigens in sufficient quantity and duration for the
transition to the development of potent adaptive
immune responses [13,41]. This hypothesis is
supported by work with Salmonella, wherein loss of
proteins under
Figure 2. S. Typhimurium dam mutants induce an attenuated inflammatory response in macrophages RAW 264.7.
Dam methylation could participate in the activation of both signalling cascades, MAPK and NFkB, in the inflammatory response induced by Salmonella spp. Then, the
expression of both NOS-2 and COX-2 and subsequently the production of NO and PGE2 is significantly reduced in cells infected with dam strains with respect to the wild
type (wt). In the same way, apoptosis in macrophages treated with Salmonella dam mutant is diminished.

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Dam function results in a number of changes in the
bacterial physiology. dam mutants appear to express
in vitro a number of genes that are normally only
produced in vivo during the initiation and progression
of bacterial infection [12,15,42]. Such patterns of
altered expression and secretion may contribute to
attenuated virulence and robust immunity observed in
vaccinated animals.

Dam and the inflammatory response in
macrophages
The ability of Salmonella dam mutants to
activate the signalling pathways of the inflammatory
response has been investigated in RAW 264.7 cells
[43] (Figure 2). The expression of NOS-2 and COX-
2, and subsequently the production of NO and PGE2,
is significantly reduced in macrophages infected with
S. Enteritidis dam strains [51]. These results are
compatible with studies showing that mice infected
with S. Typhimurium lacking the Dam protein
display reduced induction of NOS-2 and INF-γ
compared to wild type infected mice [44,45]. The
regulation of NOS-2 and COX-2 expression depend
on the activation of NF- B and alternative signalling
pathways such as those involving the ERK1/2 MAP
kinases and the family of p38 MAP kinases [46]. At
early time points after macrophage infection with
Salmonella dam mutants, the translocation of p65 to
the nucleus is notably impaired and the amount of
phosphorylated p44, p42 and p38 is clearly reduced
[43]. Taken together, these results suggest that
Salmonella activation of both signalling cascades is a
mechanism that requires Dam protein participation.
How Salmonella Dam protein participates in the
activation of macrophage signaling pathways is not
fully understood. During the course of cell invasion,
LPS activates cellular signaling pathways leading to
changes in the expression of various genes, including
those for inflammatory mediators. It is well
documented that LPS triggers, among others, the
MAPKs and NF- B pathways in macrophages
through the activation of TLR4. Interestingly, we
have found that the LPS of the S. Enteritidis dam
mutant shows a high
polysaccharide chains [Sarnacki et al., unpublished
data]. Whether this defective LPS contributes to the
attenuated inflammatory response of dam mutant-
infected macrophages is currently being investigated.
Bacterial molecules other
macrophage inflammatory response [47]. It has been
suggested that Salmonella effector proteins such as
SopB, SopE2, and certain invasion proteins may be
proportion of short
than LPS induce
involved in regulating the inflammatory response of
infected cells [47,48]. Therefore, it would be likely
that the failure of dam mutants to induce a whole
macrophage inflammatory response correlates with a
defective expression of one or more effector proteins;
previous works support this hypothesis [11,32].
Moreover, in vitro studies using epitope-tagged
(3xFLAG) strains of S. Typhimurium [49] showed
that expression and secretion of several TTSS
effector proteins are decreased in S. Typhimurium
dam mutants [38, Giacomodonato et al., unpublished
data]. Therefore, the attenuated inflammatory
response induced by dam mutants could be explained
in part to a down-regulated expression/secretion of
Salmonella effector proteins.

Conclusions
It is clear that Dam methylation is an essential
factor involved in the virulence of an increasing
number of bacterial pathogens including S. enterica.
In the near future, research studies will have to
identify genes and proteins that are differentially
expressed in response to methylation by Dam,
together with a careful characterization of regulatory
mechanism which mediate these effects in different
organisms. This information will contribute to
elucidate the different functions of Dam methylation
in the intricate regulatory networks underlying
bacterial pathogenesis. Finally, the way in which
Dam methylation participates in the control and
coordination of virulence genes expression is a
critical question that deserves further investigation.

Acknowledgments
This work was supported in part by grants from
Agencia Nacional de Promoción a la Ciencia y
Tecnología (PICT–2006-00407)
Universidad de Buenos Aires (UBACyT M608 and
M009), Argentina.

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Corresponding Author
Mónica N. Giacomodonato, PhD
Facultad de Medicina, UBA, Paraguay 2155,
(C1121ABG) Buenos Aires, Argentina.
Phone: 54-11-5950-9500
FAX: 54 11 4964 2554
Email: monicagiaco@gmail.com