Báo cáo khoa học: Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis doc

Natriuretic peptides affect Pseudomonas aeruginosa and
specifically modify lipopolysaccharide biosynthesis
Wilfried Veron
1
, Olivier Lesouhaitier
1
, Xaviera Pennanec
2
, Karine Rehel
2
, Philippe Leroux
3
,
Nicole Orange
1
and Marc G. J. Feuilloley
1
1 Laboratory of Cold Microbiology, UPRES 2123, University of Rouen, Evreux, France
2 Laboratoire de Biotechnologie et Chimie Marines, Universite
´
de Bretagne-Sud B.P. 92116, Lorient, France
3 Research Department of Microenvironment and Cell Integrated Renewal, UPRES 3829, University of Rouen, France
Bacteria of the genus Pseudomonas can adapt to a multi-
tude of environmental niches, owing to the size of their
genome and the abundance of regulatory genes [1]. The
natural resistance to b-lactams and biocides of Pseudo-
monas strains and their opportunistic behavior pose
considerable problems for hospitals [2]. Pseudomonas
aeruginosa is one of the principal microorganisms
responsible for nosocomial diseases [3]. P. aeruginosa
has considerable infectious potential, as it can infect

both surface tissues, such as skin and wounds, and inter-
nal organs, such as the lungs and urinary tract [4]. Cen-
tral nervous system infections are observed more rarely,
but such infections are steadily increasing in number
and are associated with high morbidity rates [5,6]. Con-
sistent with these clinical data, in vitro experiments have
shown that P. aeruginosa has a high specific affinity for
neurons and glial cells, and that the binding of the bac-
terium or its lipopolysaccharide (LPS) to these target
cells is associated with necrosis [7,8].
In the host, bacteria are exposed to various defense
mechanisms. Successful adaptation to an ecological
niche, such as eukaryotic tissues, clearly requires
the sensing of chemical signals, their integration and
the development of an appropriate response [9,10]. The
Keywords
cyclases; cytotoxicity; natriuretic peptides;
sensor; vfr
Correspondence
O. Lesouhaitier, Laboratory of Cold
Microbiology, UPRES 2123, University of
Rouen, 55 rue Saint Germain,
27000 Evreux, France
Fax: +33 232 29 15 55
Tel: +33 232 29 15 42
E-mail:
(Received 18 July 2007, revised 28 August
2007, accepted 18 September 2007)
doi:10.1111/j.1742-4658.2007.06109.x
Natriuretic peptides of various forms are present in animals and plants,

and display structural similarities to cyclic antibacterial peptides. Pretreat-
ment of Pseudomonas aeruginosa PAO1 with brain natriuretic peptide
(BNP) or C-type natriuretic peptide (CNP) increases bacterium-induced
glial cell necrosis. In eukaryotes, natriuretic peptides act through receptors
coupled to cyclases. We observed that stable analogs of cAMP (dibutyryl
cAMP) and cGMP (8-bromo-cGMP) mimicked the effect of brain natri-
uretic peptide and CNP on bacteria. Further evidence for the involvement
of bacterial cyclases in the regulation of P. aeruginosa PAO1 cytotoxicity
by natriuretic peptides is provided by the observed doubling of intrabacte-
rial cAMP concentration after exposure to CNP. Lipopolysaccharide (LPS)
extracted from P. aeruginosa PAO1 treated with both dibutyryl cAMP and
8-bromo-cGMP induces higher levels of necrosis than LPS extracted from
untreated bacteria. Capillary electrophoresis and MALDI-TOF MS analy-
sis have shown that differences in LPS toxicity are due to specific differ-
ences in the structure of the macromolecule. Using a strain deleted in the
vfr gene, we showed that the Vfr protein is essential for the effect of natri-
uretic peptides on P. aeruginosa PAO1 virulence. These data support the
hypothesis that P. aeruginosa has a cyclic nucleotide-dependent natriuretic
peptide sensor system that may affect virulence by activating the expression
of Vfr and LPS biosynthesis.
Abbreviations
8BcGMP, 8-bromo-cGMP; BNO, ordinary nutrient broth medium; BNP, brain natriuretic peptide; CNP, C-type natriuretic peptide; dbcAMP,
dibutyryl cAMP; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MECK, micellar electrokinetic chromatography.
5852 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS
role of the immune system in the response to Pseudo-
monas infection has been investigated in detail [10,11].
However, tissue factors or hormones, including natri-
uretic peptides, have also been described as having
antimicrobial activity [12,13]. Three types of natriuretic
peptide ) atrial natriuretic peptide, brain natriuretic

peptide (BNP) and C-type natriuretic peptide (CNP) )
were initially characterized. These molecules are major
cardiovascular and osmoregulatory factors in verte-
brates, and have been detected both in peripheral
organs, where they are produced as hormones, and in
nervous tissues, where they act as neuromodulators
[14]. Natriuretic peptides include a short loop (Fig. 1)
containing an excess of positively charged amino acids.
This structure is markedly similar to that of cationic
cyclic antimicrobial peptides [15]. The abundance of
BNP and CNP in tissues not involved in regulating
blood pressure or diuresis suggests that these neuro-
peptides probably have other activities. Natriuretic
peptides have been conserved throughout the evolution
of animals, with different forms being present in verte-
brates [16] and invertebrates [17]. Structural analogs of
animal natriuretic peptides exist in plants [18], and a
gene encoding a plant natriuretic peptide-like molecule
has recently been identified in a Gram-negative bacte-
rium [19]. Similarly, a class III cyclase with a similar
function to the GMP cyclase receptors of eukaryotic
natriuretic peptides was recently described in prokary-
otes [20]. These observations suggest that bacteria
could also employ natriuretic peptide-like molecules as
communication tools.
In the present study, we investigated the effects of
BNP and CNP (Fig. 1) on the virulence of P. aerugin-
osa PAO1. We evaluated the virulence of unexposed
bacteria and of bacteria exposed to these peptides
in vitro, using primary cultures of glial cells as the

study model. We were able to reproduce the effects of
BNP and CNP by treating the bacteria with stable
analogs of cyclic nucleotides (Fig. 1). The effects of
BNP and CNP on the intrabacterial concentrations of
cAMP and cGMP were determined, and a vfr null
mutant of P. aeruginosa PAO1 was used to investigate
the mechanism of action of these factors. Finally, the
impact of stable analogs of cyclic nucleotides on the
structure and cytotoxicity of LPS was determined by
complementary approaches.
Results
Preliminary studies showed that the treatment of
P. aeruginosa PAO1 with BNP or CNP (10
)6
m) had
no effect on bacterial growth (data not shown). The
peptides were administered at various time points, at
the start of culture or during the early part of the sta-
tionary phase. In some experiments, we added an
aliquot of peptide to the culture hourly, to take into
account possible degradation. In all cases, even on
solid medium, BNP and CNP had no effect on bacte-
rial growth or cultivability.
Effect of natriuretic peptides on the cytotoxic
activity of P. aeruginosa PAO1
The effect of BNP and CNP on the virulence of
P. aeruginosa PAO1 was studied by treating bacteria
with these peptides before mixing them with cells and
comparing the resulting cytotoxicity with that of
G

L
S
K
G
C
C
F
G
L
K
L
D
R
I
G
S
M
S
G
G
L
NH
2
-
HOOC-
hBNP
CNP
dbcAMP
8BcGMP
NH

O
N
N
N
N
O
O
O
H
H
O
O
P
O
OH
O
N
N
N
N
O
HO
H
H
O
O
P
O
OH
H

2
N
Br
H
M
V
Q
G
S
G
C
C
F
G
R
K
M
D
R
I
S
S
S
S
G
G
L
K
VLRRH
NH

2
-
HOOC-
SP
K
Fig. 1. Structure of human BNP and CNP and cyclic analogs of nu-
cleotides used in the present study. Human BNP (hBNP) is com-
posed of 32 amino acids whereas CNP contains only 22 amino
acids. dbcAMP and 8BcGMP are cell-permeable cAMP and cGMP
analogs, respectively.
W. Veron et al. Effect of natriuretic peptides on Pseudomonas
FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5853
nontreated bacteria. The model of eukaryotic cells
used here ) primary cultures of glial cells ) has been
validated in previous studies [7,8]. In all experiments,
bacteria were rinsed before exposure to glial cells, to
remove all traces of peptide. The virulence of the bac-
teria was evaluated by determining lactate dehydroge-
nase (LDH) levels in the incubation medium. LDH is
a stable enzyme present in the cytosol of eukaryotic
cells, and is released into the medium when the mem-
brane is destabilized. This destabilization is considered
to be a marker of necrosis. However, as some bacterial
strains can also generate LDH, we also checked for
the spontaneous production of this enzyme by P. aeru-
ginosa PAO1. When P. aeruginosa PAO1 was incu-
bated alone in the culture medium used for glial cells,
LDH remained undetectable (Fig. 2). In the same con-
ditions, glial cells cultured alone also released a limited
amount of LDH. The amount of LDH released in

these conditions indicates that only a small proportion
of the glial cell population (7.6 ± 1.5%; n ¼ 18) spon-
taneously underwent cell death during the course of
the experiment (6 h). This result (fewer than 10% of
the cells dying over a 6 h period) is an indicator of the
quality of the culture, and all experiments in which the
controls displayed spontaneous necrosis levels exceed-
ing this value were excluded from the analysis. When
glial cells were exposed to P. aeruginosa PAO1
(10
6
CFUÆmL
)1
, 6 h), the percentage of the cell popu-
lation displaying signs of necrosis increased to
39.0 ± 2.2%. The prior treatment of P. aeruginosa
with natriuretic peptides (10
)6
m,14h,37°C)
increased the cytotoxic activity of the bacteria signifi-
cantly. In the presence of P. aeruginosa PAO1 treated
with BNP, the percentage of the cell population
displaying signs of necrosis reached 52.4 ± 3.1%
(P<0.001). An even higher percentage was reached
(73.4 ± 4.5%, P < 0.001) when bacteria were exposed
to CNP (Fig. 2). A shorter period of prior treatment
of P. aeruginosa PAO1 with natriuretic peptides (4.5 h)
had similar effects (data not shown). CNP had a
significantly stronger effect than BNP (P<0.001;
Fig. 2). We carried out control experiments in which

glial cells were exposed only to BNP or CNP (10
)6
m).
The percentage of glial cells displaying signs of necro-
sis was identical to that of a control culture (data not
shown). Thus, the effects observed were due entirely to
the direct action of BNP or CNP on the physiology of
P. aeruginosa PAO1.
Effect of stable analogs of cAMP and cGMP on
the cytotoxic activity of P. aeruginosa PAO1
The effect of natriuretic peptides in eukaryotic cells is
mediated by different receptor subtypes with intrinsic
guanylate cyclase activity [21] or through receptors
coupled to an adenylate cyclase [22]. We therefore
investigated the effects of cell-permeable stable analogs
of cGMP [8-bromo-cGMP (8BcGMP)] and cAMP [di-
butyryl cAMP (dbcAMP)] on the virulence of P. aeru-
ginosa PAO1. As observed with natriuretic peptides,
exposure of the bacteria to dbcAMP or 8BcGMP
(10
)5
m;14h;37°C) significantly increased
(P<0.001) the potential of P. aeruginosa PAO1 to
induce necrosis in glial cells. Necrosis levels reached
57.9 ± 4.6% of the cell population when the bacteria
were exposed to dbcAMP, and 59.6 ± 5.1% when the
bacteria were first treated with 8BcGMP, versus only
39.0 ± 2.2% for nontreated P. aeruginosa PAO1
(Fig. 3). Control studies were carried out with glial
cells directly exposed to dbcAMP or 8BcGMP

(10
)5
m). The percentage of glial cells displaying signs
of necrosis was identical to that of a control culture,
indicating that the stable analogs of cyclic nucleotides
had no intrinsic effect on the viability of glial cells
(data not shown).
+
PAO1 control
-


Glial Cells
-+++
+
+
PAO1 BNP
treated
-+-
PAO1 CNP
treated
+
LDH (% of release)
0
30
50
70
90
10
Fig. 2. Cytotoxic activity of P. aeruginosa PAO1 treated with natri-

uretic peptides. Effect of BNP and CNP (10
)6
M) on the cytotoxicity
of P. aeruginosa PAO1. The cytotoxic effect of the bacterium was
determined by measuring LDH accumulation in the medium due to
the rupture of the cytoplasmic membrane of glial cells and the
release of this stable cytosolic enzyme. Values were calculated as
the mean concentration of LDH in the culture medium after 6 h of
incubation with nontreated (control) (n ¼ 47) or treated (n ¼ 24)
bacteria. Data are the means of four independent experiments.
P<0.001, significantly different.
Effect of natriuretic peptides on Pseudomonas W. Veron et al.
5854 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS
Effect of natriuretic peptides on intrabacterial
concentrations of cAMP and cGMP
As exogenous cyclic nucleotides were found to have an
effect on P. aeruginosa PAO1, we investigated the pos-
sible action of natriuretic peptides on the intrabacterial
concentrations of cAMP and cGMP. The concentra-
tion of cAMP in P. aeruginosa PAO1 grown in ordin-
ary nutrient broth medium (BNO) in the absence of
treatment was determined by ELISA. The value
obtained in early stationary phase (10.0 ± 1.4 pmolÆ
mL
)1
of bacterial extract) was used as the 100% basal
level (Fig. 4A). As we hypothesized a role for cyclic
nucleotides in the transduction of a signal mediated by
natriuretic peptides, and a rapid and early effect of
these peptides, we exposed the bacteria for a short

time (30 min) to BNP and CNP and then determined
their intracellular cAMP content. The treatment of
P. aeruginosa PAO1 with BNP (10
)6
m) did not mod-
ify cAMP levels in the bacteria (Fig. 4A). In contrast,
CNP (10
)6
m) treatment significantly increased intra-
bacterial cAMP concentrations, which reached
174.8 ± 6.3% of control values (P<0.001). The
basal concentration of cGMP in P. aeruginosa PAO1
(0.55 ± 0.16 pmolÆmL
)1
) was lower than that of
cAMP, and was at the limit of the sensitivity of the
assay. This value was used as the 100% basal level
(Fig. 4B). The exposure of P. aeruginosa PAO1 to
BNP or CNP (10
)6
m, 30 min) had no significant effect
on cGMP levels in the bacteria (Fig. 4B).
Effects of stable analogs of cAMP and cGMP on
the cytotoxicity of the LPS of P. aeruginosa PAO1
LPS is largely responsible for the cytotoxicity of Pseu-
domonas in glial cells [8]. We therefore investigated the
impact of stable analogs of cyclic nucleotides on the
endotoxic activity of this macromolecule. The cytotox-
icity of the LPS extracted from P. aeruginosa PAO1
exposed to dbcAMP or 8BcGMP (10

)5
m; 4 h; 37 °C)
was compared with that of the LPS extracted from con-
trol bacteria in the same growth phase (early stationary
phase). LPS was extracted from control and treated
bacteria, and its concentration was determined by a
2-keto-3-deoxyoctonate (KDO) assay. The mean con-
centrations of LPS measured by this technique in bacte-
rial cultures grown in the absence (0.65 mgÆmL
)1
)or
LDH (% of release)
0
10
30
70
90
50
PAO1 control
-
-

Glial Cells
+++
+
+
PAO1 dbcAMP
treated
-+-
PAO1 8BcGMP

treated
+
Fig. 3. Cytotoxic activity of P. aeruginosa PAO1 treated with cyclic
nucleotide analogs. Effect of dbcAMP and 8BcGMP (10
)5
M) on the
cytotoxicity of P. aeruginosa PAO1. Values were calculated as the
mean concentration of LDH in the culture medium after 6 h of
incubation with nontreated (control) (n ¼ 47) or treated (n ¼ 14)
bacteria. Data are the means of four independent experiments.
P<0.001, significantly different.
cAMP (% of control level)
100
200
PAO1
control
PAO1
BNP
treated treated
PAO1
CNP
PAO1
control
PAO1
BNP
treated treated
PAO1
CNP
NS
AB

0
150
50
cGMP (% of control level)
100
200
0
150
50
NS NS
Fig. 4. Intrabacterial concentration of mono-
phosphate cyclic nucleotides in P. aerugin-
osa PAO1 after exposure to natriuretic
peptides. Effect of BNP and CNP (10
)6
M)
on the intrabacterial concentration of cAMP
(A) and cGMP (B) in P. aeruginosa PAO1.
Data are the means of four independent
experiments. P<0.001, significantly differ-
ent; NS, not significantly different.
W. Veron et al. Effect of natriuretic peptides on Pseudomonas
FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5855
presence of cAMP or cGMP (0.53 mgÆmL
)1
and
0.64 mgÆmL
)1
, respectively) were in the same range. An
equivalent amount of the molecule (500 ngÆmL

)1
) was
added to the growth medium for glial cells, and its
cytotoxic activity was determined by measuring LDH
release, as previously described. The spontaneous basal
level of necrosis in glial cells was very low (4.9 ± 0.3%
of the cell population in 24 h; n ¼ 10) (Fig. 5). When
glial cells were exposed to LPS extracted from control
P. aeruginosa PAO1, 10.5 ± 0.3% of the glial cells dis-
played signs of necrosis. The necrotic potential of the
endotoxin extracted from P. aeruginosa PAO1 exposed
to dbcAMP and 8BcGMP was significantly higher,
with 13.4 ± 0.8% (P<0.001) and 15.7 ± 0.7%
(P<0.001) of the cells, respectively, displaying signs
of necrosis. Control studies were carried out with
extracts of bacterial culture medium obtained with the
same protocol used for LPS extraction and purification.
These experiments demonstrated that the effect of LPS
on glial cell viability was not due to contaminants from
the extraction buffers used to purify the endotoxin
(data not shown).
Effect of stable analogs of cAMP and cGMP on
the structure of the LPS of P. aeruginosa PAO1
The effect of stable analogs of cAMP and cGMP on
the structure of the LPS was studied using two com-
plementary techniques, micellar electrokinetic chroma-
tography (MEKC) and MALDI-TOF MS. In both
cases, LPS was extracted from P. aeruginosa PAO1,
with or without exposure to dbcAMP or 8BcGMP
(10

)5
m; 4 h; 37 °C), and purified for analysis as
described by Darveau & Hancock [23]. LPS was first
analyzed by MEKC, as this technique separates hydro-
phobic molecules differing little in size and polarity
more efficiently than HPLC. We have previously
shown that it is possible to differentiate between LPS
molecules from the same strain of Pseudomonas grown
at different temperatures by MEKC [24]. The LPS of
P. aeruginosa PAO1 grown in control conditions gave
three major peaks (Fig. 6A). The first and major peak
(peak 1) had a retention time of 8.2 min. The second
peak was associated with a series of compounds of
very similar structure and had a retention time of
11.8 min. The third major peak was well separated,
with a retention time of 22.5 min, and seemed to cor-
respond to a single molecular form that was strongly
retained and highly hydrophobic. The electrophero-
grams of LPS extracted from P. aeruginosa PAO1 trea-
ted with dbcAMP and 8BcGMP were considerably
modified (Fig. 6B,C). Peak 1, eluted close to the elec-
tro-osmotic flux, corresponded to poorly charged and
nonhydrophobic molecules ) presumably a form of
LPS with a large osidic chain and a small number of
ionized phosphate groups. The main differences
between LPS extracted from bacteria with and without
cyclic nucleotide treatment concerned the levels of
peaks 2 and 3. In dbcAMP-treated bacteria, peak 2
was much smaller and a fourth peak appeared at
17.1 min (Fig. 6B). Peak 3 was also broadened and

accompanied by secondary traces between 22.2 and
23.0 min. The difference between LPS extracted from
control and 8BcGMP-treated bacteria was even
greater. LPS from bacteria exposed to 8BcGMP dis-
played a signal corresponding to peak 1, no peak 2,
and only a small, broadened peak 3 (Fig. 6C). The
changes observed in the LPS from dbcAMP-treated
and 8BcGMP-treated bacteria suggest a decrease in
the strongly charged or very hydrophobic forms of
LPS.
MALDI-TOF MS of the LPS of P. aeruginosa
PAO1 identified a large number of compounds
(Fig. 7A). The lack of a mass spectrum library for the
LPS of P. aeruginosa PAO1 made it difficult to inter-
pret the results. A study of MS data [25–30] for
Glial Cells
LPS PAO1
LPS PAO1
control
dbcAMP treated
LPS PAO1
8BcGMP treated
LDH (% of release)
0
4
8
16
12
-
-

+++
+
+
-+-
+
Fig. 5. Cytotoxic activity of LPS from P. aeruginosa PAO1 treated
with cyclic nucleotide analogs. Effect of dbcAMP and 8BcGMP
(10
)5
M) on the cytotoxicity of the LPS from P. aeruginosa PAO1.
Values are expressed as the mean concentration of LDH in the cul-
ture medium after 24 h of incubation with LPS (500 ngÆmL
)1
) from
nontreated (control) (n ¼ 12) or treated (n ¼ 12) bacteria. Data are
the means of three independent experiments. P<0.001, signifi-
cantly different.
Effect of natriuretic peptides on Pseudomonas W. Veron et al.
5856 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS
P. aeruginosa PAO1 LPS fragments led to the identifi-
cation of four principal clusters corresponding to dif-
ferent components of the molecule. Cluster I seems to
be generated by the fragments of lipid A, whereas the
molecular masses associated with clusters II, III and
IV correspond to compounds associated with oligosac-
charide cores with different O-antigen repeating units.
The LPS extracted from P. aeruginosa PAO1 treated
with dbcAMP presented many structural variations
(Fig. 7B). In particular, the multiple peaks in the

region between 2000 and 3200 Da, corresponding to
the region of the complete oligosaccharide core with
repeated O-units [26,30], were numerous for control
LPS (Fig. 7A) and much more limited for LPS from
bacteria treated with dbcAMP (Fig. 7B). Treatment
with 8BcGMP had no obvious effect on this part of
the LPS molecule (Fig. 7C). In contrast, the profiles of
the LPS molecules from P. aeruginosa PAO1 treated
with dbcAMP and 8BcGMP generated a large number
of new compounds (36 and 18, respectively) with
molecular masses between 1000 and 1900 Da, a zone
normally attributed to the components of lipid A [25].
Effect of natriuretic peptides on the cytotoxic
activity of P. aeruginosa PAO9002
In P. aeruginosa, the protein Vfr has been shown to be
activated by both cAMP and cGMP [31]. We investi-
gated the possible involvement of Vfr in the action
of natriuretic peptides on P. aeruginosa, using strain
PAO9002, a vfr null mutant of P. aeruginosa PAO1
obtained by insertion of the accC1 gene into the vfr
site [32]. As for P. aeruginosa PAO1, we checked that
the strain did not spontaneously produce any LDH-
related molecule that might interfere with our assay
(data not shown). The basal cytotoxicity of P. aerugin-
osa PAO9002 for glial cells was intrinsically much
higher than that of P. aeruginosa PAO1. We therefore
reduced the time for which glial cells were incubated
with P. aeruginosa PAO9002 to 2.5 h. In these condi-
tions, the percentage of cells displaying signs of necro-
sis following the exposure of glial cells to P. aeruginosa

PAO9002 (10
6
CFUÆmL
)1
) was in a similar range to
that following the exposure of cells to P. aeruginosa
PAO1, i.e. 39.0 ± 2.2%. In contrast to the results
obtained with P. aeruginosa PAO1, the prior treatment
of P. aeruginosa PAO9002 with BNP or CNP (10
)6
m;
14 h; 37 °C) had no significant effect on the cytotoxic
potential of this strain (Fig. 8).
Cytotoxicity and chemical properties of the LPS
extracted from P. aeruginosa PAO9002
The LPS of P. aeruginosa PAO9002 was extracted and
purified, and its cytotoxicity was determined in pri-
mary cultures of glial cells as described above. The
LPS extracted from P. aeruginosa PAO9002 was less
cytotoxic than that extracted from P. aeruginosa PAO1
PAO1
control
PAO1
dbcAMP
treated
PAO1
8BcGMP
treated
Retention time (min)
Absorbance Units (203 nm)

0102030
0.2
0.1
0
0.2
0.1
0
0.2
0.1
0
2
3
A
B
C
4
EOF
1
Fig. 6. MECK analysis of LPS treated with cyclic nucleotide ana-
logs. MECK analysis of the LPS extracted from control (A),
dbcAMP (10
)5
M)-treated (B) or 8BcGMP (10
)5
M)-treated (C)
P. aeruginosa PAO1. EOF, electro-osmotic flux. Arrows and num-
bers refer to the different molecular forms identified in the LPS
from P. aeruginosa PAO1.
W. Veron et al. Effect of natriuretic peptides on Pseudomonas
FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5857

(Fig. 9A). The percentage of the cell population under-
going necrosis in the presence of LPS from P. aerug-
inosa PAO9002 (14.56 ± 0.98%) was higher than
that normally measured in a control culture
(9.02 ± 0.24%), but significantly lower (P<0.01)
than that in cultures exposed to LPS from P. aerugin-
osa PAO1 (18.05 ± 0.42%). MEKC analysis of this
LPS revealed marked differences from the LPS
extracted from P. aeruginosa PAO1 (Fig. 9B). Indeed,
the diversity of molecular forms identified in the LPS
from P. aeruginosa PAO9002 was very low. Most of
the signal was concentrated in peak 1. Another form,
denoted peak 5, appeared at 20.3 min. A small signal
was also detected around peak 2. The electrophero-
gram profile of the LPS from P. aeruginosa PAO9002
closely resembled that of the LPS from P. aeruginosa
PAO1 after treatment with 8BcGMP.
Discussion
The structure of BNP and CNP and the results pub-
lished by Krause et al. [13] suggested that human
2777.91
A
B
C
6456.87
7212.73
8367.22
0.00
0.25
0.50

0.75
1.00
1.25
X10
4
X10
4
X10
4
Intens. [a.u.]Intens. [a.u.]Intens. [a.u.]
2000 4000 6000 8000 10000 12000 14000 16000 18000
m/z
2000 4000 6000 8000 10000 12000 14000 16000 18000
m/z
2000 4000 6000 8000 10000 12000 14000 16000 18000
m/z
3062.49
6458.10
7317.12
0.0
0.5
1.0
1.5
2.0
2.5
3.0
2778.75
6458.12
5337.87
12165.87

0
1
2
3
4
LPS PAO1 control
LPS PAO1 dbcAMP treated
LPS PAO1 8BcGMP treated
I
II
III
IV
Fig. 7. MALDI-TOF analysis of LPS treated with cyclic nucleotide analogs. MALDI-TOF mass spectra obtained from the LPS extracted from
control (A), dbcAMP (10
)5
M)-treated (B) or 8BcGMP (10
)5
M)-treated (C) P. aeruginosa PAO1. Roman numerals (A) indicate clusters of peaks
corresponding to compounds with similar molecular masses.
Effect of natriuretic peptides on Pseudomonas W. Veron et al.
5858 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS
natriuretic peptides, and not rat peptides, might have
antibacterial activity. Our preliminary studies showed
that, in contrast, at micromolar concentrations similar
to those encountered in animal tissues, BNP and CNP
have no antibacterial effect. As antibacterial agents
are also known to have indirect effects on bacterial
virulence [33], we used a well-characterized in vitro
assay developed in our laboratory [7,8] to investigate
the effects of BNP and CNP on the cytotoxicity of

bacteria.
We cultured P. aeruginosa in the presence of BNP
or CNP. Bacteria were exposed to the peptides during
the exponential growth phase and were then rinsed to
remove any trace of BNP or CNP before mixing with
primary cultures of glial cells. As confirmed by con-
trols, in which natriuretic peptides were directly mixed
with glial cells, our results can only be explained by
changes in bacterial virulence. The observation that
treating P. aeruginosa with BNP and CNP increases
the cytotoxic effect of the bacteria suggests that natri-
uretic factors can affect the physiology of microorgan-
isms sufficiently to modify virulence factor production,
but not enough to affect the growth and survival of
P. aeruginosa. These results are entirely consistent with
published data suggesting that antimicrobial peptides
can exert multiple effects, not simply inhibiting bacte-
rial growth, but also interfering with microbial physiol-
ogy, including the capacity to produce virulence
factors or even toxin activity [33].
Peptides cannot penetrate cells freely. Thus, in
eukaryotic cells, natriuretic peptides exert their effects
through three different cytoplasmic membrane receptor
subtypes with intrinsic guanylate cyclase activity [21]
or indirectly coupled to an adenylate cyclase [22]. As
in investigations of the possible involvement of cyclic
nucleotide phosphates in the mechanism of action of
neurohormones in eukaryotes [34], we tested the effect
of stable analogs of cGMP and cAMP on P. aerugin-
osa. The exposure of bacteria to 8BcGMP and

dbcAMP fully reproduced the effects of BNP and
CNP, increasing necrosis. As exogenous cyclic nucleo-
tides affected P. aeruginosa PAO1 virulence, we inves-
tigated the possible effects of natriuretic peptides on
intrabacterial cAMP and cGMP concentrations. The
level of cAMP in P. aeruginosa was unaffected by
exposure to BNP. In contrast, CNP induced a marked
LDH (% of release)
10
20
AB
Glial
cells
LPS
PAO1
LPS
PAO9002
0
15
5
1
25
0.2
0.1
0
Absorbance Units (203 nm)
EOF
Retention time (min)
0 102030
Fig. 9. Characteristics of the LPS extracted

from the vfr null mutant P. aeruginosa
PAO9002. Cytotoxic activity (A) and MEKC
electropherogram (B) of the LPS extracted
from the vfr null mutant of P. aeruginosa
PAO9002. Cytotoxicity values were calcu-
lated as the means of three independent
experiments. P<0.001, significantly differ-
ent; P<0.01, significantly different; EOF,
electro-osmotic flux. Arrows and numbers
refer to the different molecular forms identi-
fied in the LPS from P. aeruginosa
PAO9002.
PAO9002
control
-
PAO9002 BNP
treated
PAO9002 CNP
treated
-
+
-
+
-
+
-
-
LDH (% of release)
0
30

40
50
20
NS NS
10
Fig. 8. Cytotoxic activity of P. aeruginosa PAO9002 treated with
natriuretic peptides. Effect of BNP and CNP (10
)6
M) on the cyto-
toxicity of P. aeruginosa PAO9002. Values were calculated as the
mean concentration of LDH in the culture medium after 2.5 h of
incubation with nontreated (control) (n ¼ 16) or treated (n ¼ 16)
bacteria. Data are the means of four independent experiments. NS,
not significantly different.
W. Veron et al. Effect of natriuretic peptides on Pseudomonas
FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5859
increase in cAMP concentration. The basal concentra-
tion of cGMP in P. aeruginosa was very low, at the
limit of the sensitivity of the assay. We observed only
a slight, nonsignificant increase in cGMP concentra-
tion in bacteria treated with CNP. We cannot exclude
the possibility of an increase in cyclic nucleotide con-
centrations due to mechanisms involving a membrane
phosphotransferase system and catabolic repression
[35], but the similarity of the effects of natriuretic pep-
tides in eukaryotes and prokaryotes and the growing
number of adenylyl and guanylyl cyclases identified in
bacteria [36] are consistent with the involvement of
bacterial cyclases in the response of P. aeruginosa to
natriuretic peptides. Consistent with these results, it

appears that in addition to the well-characterized bac-
terial secondary messenger, cyclic di-GMP, levels of
cyclic monophosphate nucleotides probably also play a
crucial role in integrating the environmental signals
transmitted to the bacterial cell surface [37].
In Pseudomonas, as in all other Gram-negative bac-
teria, the endotoxin is a major virulence factor
released on the death of the bacterium, but also pro-
duced in vesicular forms as an offensive weapon,
particularly in response to antimicrobial molecules
[38,39]. We investigated the possible role of LPS in
the response of P. aeruginosa to natriuretic peptides,
by studying the impact of stable analogs of cAMP
and cGMP on the cytotoxic activity and chemical
properties of the LPS from P. aeruginosa PAO1. We
used cyclic nucleotides rather than the natriuretic pep-
tides themselves, because of technical limitations, as
large volumes of bacterial culture were required, and
the correspondingly large quantities of peptide
required would have been unreasonably expensive.
P. aeruginosa is known to respond rapidly to stress
and to antimicrobial drugs by modulating the struc-
ture of its LPS [40]. P. aeruginosa should react simi-
larly to natriuretic peptides or stable analogs of cyclic
nucleotides. This hypothesis is supported by the
results of MEKC analysis. Indeed, in bacteria treated
with cyclic nucleotides, the greater cytotoxicity of the
LPS appeared to be associated with a decrease in
expression of the strongly charged or very hydropho-
bic forms of LPS by P. aeruginosa. This change was

particularly marked in 8BcGMP-treated bacteria, in
which an overall decrease in the diversity of LPS iso-
forms was observed. In the absence of a mass spec-
trum database for P. aeruginosa PAO1 LPS, the
complexity of this molecule made it very difficult to
interpret the spectra obtained by MALDI-TOF MS.
However, many essential subfragments of the LPS
were found to have been modified in bacteria treated
with dbcAMP and 8BcGMP. The more obvious
changes concerned the 1200–2050 Da zone, in which
new peaks appeared, with molecules of masses
between 2200 and 3100 Da tending to disappear.
Compounds with a molecular mass of around 1300–
1500 Da are generally considered to be signature
components of lipid A [25], and molecules of 2500–
2900 Da are considered to be fragments of the oligo-
saccharide core with repeat O-units [26]. We can
therefore speculate that treatment with dbcAMP and
8BcGMP induces the reorganization of both the
hydrophobic and polar regions of LPS, leading to an
increase in the heterogeneity or number of acylated
components of lipid A and a simplification or
decrease in the length of the oligosaccharide core. As
lipid A plays a large part in the toxicity of LPS
[41,42], these modifications may account for the
greater cytotoxicity of the LPS extracted from
dbcAMP-treated and 8BcGMP-treated bacteria.
We investigated the possible involvement of cyclic
nucleotides in the action of natriuretic peptides in
P. aeruginosa in more detail by carrying out the same

series of experiments with P. aeruginosa PAO9002, a
vfr null mutant of P. aeruginosa PAO1 [32]. The pro-
tein Vfr is a cAMP-binding protein that controls the
production of many virulence factors [43,44]. The basal
cytotoxicity of P. aeruginosa PAO9002 was particularly
high, and this strain appeared to be totally insensitive
to BNP and CNP. The very high virulence of P. aeru-
ginosa PAO9002 suggests that the knock-down of vfr
fixes the bacterium in a maximum virulence configura-
tion, in which the bacterium cannot respond to natri-
uretic peptides. This confirms the importance of cAMP
in the mechanism of action of natriuretic peptides in
P. aeruginosa. In this species, Vfr may be activated by
both cAMP and cGMP, and is unlikely to discriminate
between the two types of cyclic nucleotide [31]. This
may account for the identical effects of stable analogs
of cAMP and cGMP in P. aeruginosa PAO1. This
hypothesis is also entirely consistent with the results of
MEKC analysis, showing that the LPS of P. aerugin-
osa PAO9002 is very similar to that of P. aeruginosa
PAO1 treated with 8BcGMP.
This study demonstrates, for the first time, that
natriuretic peptides can modulate the virulence of
P. aeruginosa. Our data strongly suggest that the
action of BNP and CNP in the bacteria is linked to
cyclic nucleotide production and the induction of Vfr
protein activation. The signal transduction cascade
generated by this mechanism should regulate bacterial
virulence, at least partly by modulating LPS structure.
Our results suggest that P. aeruginosa has a mem-

brane-associated natriuretic peptide sensor, and open
up new areas of research into the evolution of the
Effect of natriuretic peptides on Pseudomonas W. Veron et al.
5860 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS
physiological role of natriuretic peptides. This discov-
ery also provides an opportunity for the development
of new therapeutic agents.
Experimental procedures
Reagents and test substances
DMEM and Ham’s F12 culture medium, Hepes buffer,
poly(l-lysine), insulin, dbcAMP, 8BcGMP and human
BNP were purchased from Sigma-Aldrich (St Quentin Fal-
lavier, France). CNP peptide was obtained from Neosystem
(Strasbourg, France). a-Cyano-4-hydroxycinnamic acid,
trifluoroacetic acid and molecules for MS calibration were
obtained from Sigma-Aldrich Corp. (St Louis, MO, USA).
Acetonitrile was purchased from Fisher Scientific (Lough-
borough, UK). Fetal bovine serum, l-glutamine and anti-
biotic ⁄ antimycotic solutions were obtained from Cambrex
(Emerainville, France). The Cytotox 96 kit was purchased
from Promega (Charbonnie
`
res, France).
Bacterial cultures
P. aeruginosa PAO1 was obtained from an international
collection. The vfr null mutant of P. aeruginosa PAO1,
P. aeruginosa 9002, was generously provided by S. J. Suh
(University of Wisconsin-Madison, WI, USA). Both strains
were grown in BNO (Merck, Darmstadt, Germany) at
37 °C. For treatment with natriuretic peptides or cyclic

nucleotides, bacteria were transferred to 10 mL of BNO
containing (or not containing in the case of the control) the
test substances, and were cultured overnight until the start
of stationary phase. Just before the infection assays, bacteria
in early stationary phase were harvested by centrifugation in
an Eppendorf centrifuge tube (2500 g, 4 min, 20 °C) and
resuspended at a cell density of 10
6
CFUÆmL
)1
in glial cell
culture medium without antibiotics and antimycotics. The
density of the bacterial suspension was determined by
absorption at 580 nm, using a spectrophotometer (Thermo-
Spectronics, Cambridge, UK). Bacterial density and the
absence of contamination were checked by plating.
Animals
Adult Wistar rats (180–200 g) were purchased from a com-
mercial source (De
´
pre
´
, St Doulchard, France). Newborn
Wistar rats were obtained by mating in the laboratory.
Animals were housed in an animal house (Agreement
no. AGEXP27.01) in which the temperature (20 °C) and
photoperiod (12 h day ⁄ 12 h night) were artificially con-
trolled. Animal manipulations were performed under the
supervision of authorized investigators and according to the
European Communities Council Directive of 24 November

1986 (86 ⁄ 609 ⁄ EEC).
Glial cell culture
Newborn rats were decapitated 48–72 h after birth, in ster-
ile conditions. The brain was quickly extracted and rinsed
in glial culture medium consisting of DMEM ⁄ Ham’s med-
ium (2 : 1) supplemented with 10% fetal bovine serum,
2mm glutamine, 0.001% insulin, 5 mm Hepes, 0.3% glu-
cose and 1% antibiotic ⁄ antimycotic solution. The meninges
were removed, and the telencephalon was carefully dis-
sected, immersed in glial culture medium, and mechanically
dispersed for 5 min by gentle aspiration through a sterile
needle. The suspension was filtered through a sterile nylon
filter with 82 lm pores to remove the remaining tissue frag-
ments. Cells were counted and layered, at a concentration
of 10
5
cells per slide, on glass slides coated with 50 lgÆmL
)1
poly(l-lysine). Glial cells were incubated at 37 °Cina
humidified atmosphere containing 5% CO
2
and were
allowed to grow for 9–14 days, to obtain a confluent cul-
ture in all culture wells. The culture medium was changed
the day after plating and every 2 days thereafter.
Measurement of the release of cytosolic LDH by
glial cells
LDH is a stable cytosolic enzyme released into the culture
medium upon cell lysis. Its use as an indicator of necrosis in
glial cells has been validated [8]. The amount of LDH released

by eukaryotic cells incubated with bacteria was determined
with the Cytotox 96 enzymatic assay (Promega). Glial cells
were incubated for 6 h with control or treated P. aeruginosa
PAO1 at a concentration of 10
6
CFUÆmL
)1
. For studies with
P. aeruginosa PAO9002, the incubation time was reduced to
2.5 h, because this strain is highly cytotoxic. A lysis buffer,
consisting of 9% Triton X-100 in water, was used to deter-
mine the maximum amount of LDH released by glial cells in
our experimental conditions (100% LDH release). A back-
ground level, corresponding to 0% LDH release, was estab-
lished using culture medium alone, making it possible to
subtract the contribution to LDH activity of the medium used
for glial cell culture. The percentage of LDH release in the cell
population was then determined by the equation:
% LDH ¼ðD sample À D 0%ÞÂ100=
ðD 100% À D 0%Þ
where D is attenuance at 490 nm.
The assay was sensitive enough for determination of a
concentration of LDH equivalent to the lysis of 1% of the
cell population.
Measurement of intrabacterial cAMP and cGMP
concentrations
The levels of cAMP and cGMP in bacteria exposed to
natriuretic peptides were determined using cAMP and a
W. Veron et al. Effect of natriuretic peptides on Pseudomonas
FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5861

cGMP direct enzyme immunoassay kits from Sigma-
Aldrich. P. aeruginosa (10
6
CFUÆmL
)1
) in early station-
ary phase was incubated for 30 min at 37 °Cin10mL
of BNO supplemented with BNP or CNP (10
)6
m). Bac-
teria were then centrifuged (6 min; 2500 g), and the
pellets were resuspended in saline. The samples were
boiled for 10 min, ultracentrifuged (1 h; 62 000 g)to
remove cell debris, and freeze-dried. The resulting resi-
dues were resuspended in 1 mL of water and assayed
for cAMP or cGMP, according to the kit supplier’s
protocol.
LPS extraction and purification
LPS was purified from P. aeruginosa PAO1 according to a
procedure based on that of Darveau & Hancock [23].
Briefly, bacteria in early stationary phase were harvested by
centrifugation (6000 g, 10 min, 20 °C). P. aeruginosa PAO1
pellets were suspended in 10 mm Tris buffer containing
2mm MgCl
2
, 200 lgÆmL
)1
pancreatic DNase and
50 lgÆmL
)1

pancreatic RNase, and were submitted to four
bursts of sonication of 30 s at a probe density of 70. The
suspension was then incubated for 2 h at 37 °C. After this
period, tetrasodium-EDTA, Tween-20 and Tris ⁄ HCl were
added. The sample was centrifuged (10 000 g, 30 min,
20 °C) to remove peptidoglycan. The supernatant was incu-
bated overnight with protease, at 37 °C, with constant
shaking. Two volumes of 0.375 m MgCl
2
in 95% ethanol
were added, and the mixture was cooled to 0 °C. The sam-
ple was then centrifuged (12 000 g, 15 min, 0 °C), and the
pellet was sonicated in a solution of Tween-20 and tetraso-
dium-EDTA. The pH of the solution was lowered to 7, to
prevent lipid saponification. The solution was incubated for
30 min at 85 °C, to ensure that outer membrane proteins
were denatured, and the pH of the solution was increased
to 9.5. Protease was then added, and the sample was incu-
bated overnight at 37 °C. Two volumes of 0.375 m MgCl
2
in 95% ethanol were added, and the sample was centri-
fuged (12 000 g, 15min, 0 °C). The pellet was resuspended
in 10 mm Tris ⁄ HCl, sonicated, and centrifuged twice to
remove insoluble Mg
2+
-EDTA crystals. The supernatant
was then ultracentrifuged (62 000 g, 2 h, 15 °C), and the
pellet, which contained LPS, was resuspended in distilled
water and used for the KDO assay. The control extraction
buffer was prepared following the same procedure, but

starting with sterile BNO.
Capillary electrophoresis of LPS
Purified bacterial LPS molecules were analyzed by MEKC,
using a Beckman (Gagny, France) P ⁄ ACE 5510 system
equipped with a diode array detector (detection range, 190–
600 nm; wavelength of the curves presented, k ¼ 203 nm)
and refrigerated injection system. A fused-silica capillary
tube (50 lm internal diameter · 57 cm; 50 cm to detector)
was purchased from Beckman Coulter (Villepinte, France).
The capillary was installed in a refrigerated cartridge and
conditioned with 1 m NaOH for 4 h before rinsing and
equilibration with running buffer. The separation buffer for
MEKC consisted of 25 mm boric acid (pH 9) supplemented
with SDS (80 mm). The potential used for separation
(25 kV) was determined as the limit of linearity of the curve
for the current–voltage relationships. Samples were injected
by N
2
hydrostatic pressure (0.5 lbÆin
)2
, 20 s) and analyzed
over a 30 min period.
MALDI-TOF MS analysis of LPS
For each test, 1 lL of purified LPS was carefully spotted
onto each target well and allowed to dry in air. A solution
of a-cyano-4-hydroxycinnamic acid matrix (14 mgÆmL
)1
)
was prepared in acetronitrile ⁄ 2.5% trifluoroacetic acid
(1 : 1, v ⁄ v). The matrix was sonicated for 30 min, and 1 lL

of matrix solution was added to each well and allowed to
dry at room temperature. MS was performed in linear
mode on a Microflex Time-of-Flight machine (Bruker Dal-
tonik GmbH, Leipzig, Germany) with a pulsed ultraviolet
laser at 337 nm. The machine was run in positive mode and
calibrated with 1 lL of a 10 pm protein standard mixture
of angiotensin II, adrenocorticotrophic hormone Clip (18–
39), human insulin, oxidized B-chain, bovine insulin and
cytochrome c spotted onto the target plate. Each final spec-
trum consisted of 200 laser shots.
Statistics
Assay results were determined for a minimum of 10 sample
measurements, from three or four independent prepara-
tions. Student’s t-test was used to compare the means of
values within sets of experiments.
Acknowledgements
We wish to thank Dr Suh SJ (Auburn, USA) for gen-
erously providing the P. aeruginosa 9002 strain. This
work was supported by grants from the Conseil Ge
´
ne
´
-
ral de l’Eure and the Communaute
´
d’Agglome
´
ration
d’Evreux.
References

1 Goodman AL & Lory S (2004) Analysis of regulatory
networks in Pseudomonas aeruginosa by genomewide
transcriptional profiling. Curr Opin Microbiol 7, 39–44.
2 Clark NM, Patterson J & Lynch JP (2003) Antimicro-
bial resistance among Gram-negative organisms in the
intensive care unit. Curr Opin Crit Care 9, 413–423.
3 Richards MJ, Edwards JR, Culver DH & Gaynes RP
(2000) Nosocomial infections in combined medical–
Effect of natriuretic peptides on Pseudomonas W. Veron et al.
5862 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS
surgical intensive care units in the United States. Infect
Control Hosp Epidemiol 21, 510–515.
4 Bagshaw SM & Laupland KB (2006) Epidemiology of
intensive care unit-acquired urinary tract infections.
Curr Opin Infect Dis 19, 67–71.
5 Feuilloley MGJ, Mezghani-Abdelmoula S, Picot L,
Lesouhaitier O, Merieau A, Guerillon J, Boujedaı
¨
ni N,
Cazin L & Orange N (2003) Involvement of Pseudomo-
nas and related species in central nervous system infec-
tions. Recent Adv Dev Microbiol 7, 55–71.
6 Lu CH, Chang WN, Chuang YC & Chang HW (1999)
Gram-negative bacillary meningitis in adult post-neuro-
surgical patients. Surg Neurol 52, 438–444.
7 Picot L, Mezghani-Abdelmoula S, Merieau A, Leroux
P, Cazin L, Orange N & Feuilloley MGJ (2001) Pseudo-
monas fluorescens as a potential pathogen: adherence to
nerve cells. Microb Infect 3, 985–995.
8 Picot L, Chevalier S, Mezghani-Abdelmoula S, Merieau

A, Lesouhaitier O, Leroux P, Cazin L, Orange N &
Feuilloley MGJ (2003) Cytotoxic effects of the lipopoly-
saccharide from Pseudomonas fluorescens on neurons
and glial cells. Microb Pathogen 35, 95–106.
9 Camilli A & Bassler BL (2006) Bacterial small-molecule
signaling pathways. Science 311, 1113–1116.
10 Wu L, Estrada O, Zaborina O, Bains M, Shen L, Koh-
ler JE, Patel N, Musch MW, Chang EB, Fu YX et al.
(2005) Recognition of host immune activation by Pseu-
domonas aeruginosa. Science 309, 774–777.
11 Ciofu O (2003) Pseudomonas aeruginosa chromosomal
beta-lactamase in patients with cystic fibrosis and
chronic lung infection. Mechanism of antibiotic resis-
tance and target of the humoral immune response.
APMIS Suppl 116, 1–47.
12 Metz-Boutigue MH, Kieffer AE, Goumon Y & Aunis
D (2003) Innate immunity: involvement of new neuro-
peptides. Trends Microbiol 11, 585–592.
13 Krause A, Liepke C, Meyer M, Adermann K, Forss-
mann WG & Maronde E (2001) Human natriuretic pep-
tides exhibit antimicrobial activity. Eur J Med Res 6,
215–218.
14 Jamison RL, Canaan-Kuhl S & Pratt R (1992) The
natriuretic peptides and their receptors. Am J Kidney
Dis 20, 519–530.
15 Bulet P, Sto
¨
cklin R & Menin L (2004) Anti-microbial
peptides: from invertebrates to vertebrates. Immunol Rev
198, 169–184.

16 Inoue K, Nasure K, Yamagami S, Mitani H, Suzuki N
& Takei Y (2003) Four functionally distinct C-type
natriuretic peptides found in fish reveal evolutionary
history of the natriuretic system. Proc Natl Acad Sci
USA 100, 10079–10084.
17 Poulos JE, Gower WR, Friedl FE & Vesely DL
(1995) Atrial natriuretic peptide gene expression within
invertebrate hearts. Genl Comp Endocrinol 100, 61–
68.
18 Gehring CA & Irving HK (2003) Natriuretic peptides
)
a class of heterologous molecules in plants. Int J
Biochem Cell Biol 35, 1318–1322.
19 Nembaware V, Seoighe C, Sayed M & Gehring C
(2004) A plant natriuretic peptide-like gene in the bacte-
rial pathogen Xanthomonas axonopodis may induce
hyper-hydration in the plant host: a hypothesis of
molecular mimicry. BMC Evol Biol 24, 10–12.
20 Kasahara M, Unno T, Yashiro K & Ohmori M (2001)
CyaG, a novel cyanobacterial adenylyl cyclase and a
possible ancestor of mammalian guanylyl cyclase. J Biol
Chem 276, 10564–10569.
21 Potter LR & Hunter T (2001) Guanylyl cyclase-linked
natriuretic peptide receptors: structure and regulation.
J Biol Chem 276, 6057–6060.
22 Anand-Srivastava MB, Sehl PD & Lowe DG (1996)
Cytoplasmic domain of natriuretic peptide receptor-C
inhibits adenylyl cyclase. Involvement of a pertussis
toxin-sensitive G protein. J Biol Chem 271, 19324–19329.
23 Darveau RP & Hancock RE (1983) Procedure for isola-

tion of bacterial lipopolysaccharides from both smooth
and rough Pseudomonas aeruginosa and Salmonella
typhimurium strains. J Bacteriol 155, 831–838.
24 Picot L, Chevalier S, Abdelmoula S, Meriau A, Lesou-
haitier O, Guerillon J, Leroux P, Cazin L, Orange N &
Feuilloley MGJ (2004) Regulation of the cytotoxic
effects of Pseudomonas fluorescens by growth tempera-
ture. Res Microbiol 155, 39–46.
25 Bedoux G, Valle
´
e-Re
´
hel K, Kooistra O, Za
¨
hringer U &
Haras D (2004) Lipid A components from Pseudomonas
aeruginosa PAO1 (serotype O5) and mutant strains
investigated by electrospray ionization ion-trap mass
spectrometry. J Mass Spectrom 39, 505–513.
26 Bystrova OV, Knirel YA, Lindner B, Kocharova NA,
Kondakova AN, Za
¨
hringer U & Pier GB (2006) Struc-
tures of the core oligosaccharide and O-units in the
R- and SR-type lipopolysaccharides of reference strains
of Pseudomonas aeruginosa O-serogroups. FEMS Immu-
nol Med Microbiol 46, 85–99.
27 Knirel YA, Bystrova OV, Shashkov AS, Kocharova
NA, Senchenkova SN, Moll H, Lindner B, Za
¨

hringer
U, Hatano K & Pier GB (2001) Structural analysis of
the lipoplysaccharide core of a rough, cystic fibrosis iso-
late of Pseudomonas aeruginosa. Eur J Biochem 268,
4708–4719.
28 Kooistra O, Bedoux G, Brecker L, Lindner B, Sanchez-
Carballo P, Haras D & Za
¨
hringer U (2003) Structure of
a highly phosphorylated lipopolysaccharide core in the
DalgC mutants derived from Pseudomonas aeruginosa
wild-type strains PAO1 (serogroup O5) and PAC1R
(serogroup O3). Carbohydr Res 338, 2667–2677.
29 Sadovskaya I, Brisson JR, Lam JS, Richards JC & Alt-
man E (1998) Structural elucidation of the lipopolysac-
charide core regions of the wild-type strain PAO1 and
O-chain-deficient mutant strains AK1401 and AK1012
W. Veron et al. Effect of natriuretic peptides on Pseudomonas
FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS 5863
from Pseudomonas aeruginosa serotype O5. Eur J Bio-
chem 255, 673–684.
30 Sadovskaya I, Brisson JR, Thibault P, Richards JC,
Lam JS & Altman E (2000) Structural characterization
of the outer core and O-chain linkage region of lipo-
polysaccharide from Pseudomonas aeruginosa serotype
O5. Eur J Biochem 267, 1640–1650.
31 Beatson SA, Whitchurch CB, Sargent JL, Levesque RC
& Mattick JS (2002) Differential regulation of twitching
motility and elastase production by Vfr in Pseudomonas
aeruginosa. J Bacteriol 184, 3605–3613.

32 Suh SJ, Runyen-Janecky LJ, Maleniak TC, Hager P,
MacGregor CH, Zielinski-Mosny NA, Phibbs PV Jr &
West SEH (2002) Effect of vfr mutation on global gene
expression and catabolite repression control of Pseudo-
monas aeruginosa. Microbiology 148, 1561–1569.
33 Hancock REW & Scott MG (2000) The role of antimi-
crobial peptides in animal defenses. Proc Natl Acad Sci
USA 97, 8856–8861.
34 Feuilloley M & Vaudry H (1996) Role of the cytoskele-
ton in adrenocortical cells. Endocr Rev 17 , 269–288.
35 Saier MH Jr (1989) Protein phosphorylation and allo-
steric control of inducer exclusion and catabolite repres-
sion by the bacterial phosphoenolpyruvate: sugar
phosphotransferase system. Microbiol Rev 53, 109–120.
36 Baker DA & Kelly JM (2004) Structure, function and
evolution of microbial adenylyl and guanylyl cyclases.
Mol Microbiol 52, 1229–1242.
37 D’Argenio DA & Miller SI (2004) Cyclic di-GMP as a
bacterial second messenger. Microbiology 150, 2497–
2502.
38 Kadurugamuwa JL & Beveridge TJ (1995) Virulence
factors are released from Pseudomonas aeruginosa in
association with membrane vesicles during normal
growth and exposure to gentamicin: a novel
mechanism of enzyme secretion. J Bacteriol 177,
3998–4008.
39 McPhee JB, Lewenza S & Hancock REW (2003) Cat-
ionic antimicrobial peptides activate a two-component
regulatory system, PmrA–PmrB, that regulates resis-
tance to polymyxin B and cationic antimicrobial pep-

tides in Pseudomonas aeruginosa. Mol Microbiol 50,
205–217.
40 Kadurugamuwa JL, Lam JS & Beveridge TJ (1993)
Interaction of gentamicin with the A band and B band
lipopolysaccharides of Pseudomonas aeruginosa and its
possible lethal effect. Antimicrob Agents Chemother 37,
715–721.
41 Ernst RK, Hajjar AM, Tsai JH, Moskowitz SM, Wilson
CB & Miller SI (2003) Pseudomonas aeruginosa lipid A
diversity and its recognition by Toll-like receptor 4.
J Endotoxin Res 9, 395–400.
42 Pier GB (2007) Pseudomonas aeruginosa lipopolysaccha-
ride: a major virulence factor, initiator of inflammation
and target for effective immunity. Int J Med Microbiol
297, 277–295.
43 Whitchurch CB, Beatson SA, Comolli JC, Jakobsen T,
Sargent JL, Bertrand JJ, West J, Klausen M, Whaite
LL, Kang PJ et al. (2005) Pseudomonas aeruginosa fimL
regulates multiple virulence functions by intersecting
with vfr-modulated pathways. Mol Microbiol 55, 1357–
1378.
44 Wolfgang MC, Lee VT, Gilmore E & Lory S (2003)
Coordinate regulation of bacterial virulence genes by a
novel adenylate cyclase-dependent signaling pathway.
Dev Cell 4, 253–263.
Effect of natriuretic peptides on Pseudomonas W. Veron et al.
5864 FEBS Journal 274 (2007) 5852–5864 ª 2007 The Authors Journal compilation ª 2007 FEBS