aeruginosa PAO1 cytotoxicity by natriuretic peptides is provided by the observed doubling of intrabacte-rial cAMP concentration after exposure to CNP.. As observed with natriuretic pepti
Trang 1Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis
Wilfried Veron1, Olivier Lesouhaitier1, Xaviera Pennanec2, Karine Rehel2, Philippe Leroux3,
Nicole Orange1and Marc G J Feuilloley1
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: olivier.lesouhait@univ-rouen.fr
(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.
Trang 2role of the immune system in the response to
Pseudo-monasinfection 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 inverteverte-brates [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)6m) 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
O O P
O
N
N N
N
O HO
O O P
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 V L R R H
NH 2
-
HOOC-S P 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.
Trang 3nontreated 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%; n¼ 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 (106CFUÆmL)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)6m, 14 h, 37C) 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)6m) 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 aeruginosaPAO1
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)5m; 14 h; 37C) 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)5m) 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
-PAO1 CNP
treated - - - - +
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)6M ) 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.
Trang 4Effect 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)1of 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)6m) did not mod-ify cAMP levels in the bacteria (Fig 4A) In contrast, CNP (10)6m) 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)6m, 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-domonasin 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)5m; 4 h; 37C) 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
0
10
30
70
90
50
PAO1 control
+ +
PAO1 dbcAMP
-PAO1 8BcGMP
Fig 3 Cytotoxic activity of P aeruginosa PAO1 treated with cyclic
nucleotide analogs Effect of dbcAMP and 8BcGMP (10)5M ) 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.
100 200
PAO1 control
PAO1 BNP treated treated
PAO1 CNP
PAO1 control
PAO1 BNP treated treated
PAO1 CNP
NS
0
150
50
100 200
0
150
50
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)6M )
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.
Trang 5presence 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; n¼ 10) (Fig 5) When
glial cells were exposed to LPS extracted from control
P aeruginosaPAO1, 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)5m; 4 h; 37C), 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
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)5M ) 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.
Trang 6P aeruginosaPAO1 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 (106CFUÆmL)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)6m;
14 h; 37C) 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)
0.2
0.1
0
0.2
0.1
0
0.2
0.1
0
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)5M )-treated (B) or 8BcGMP (10)5M )-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.
Trang 7(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
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)5M )-treated (B) or 8BcGMP (10)5M )-treated (C) P aeruginosa PAO1 Roman numerals (A) indicate clusters of peaks corresponding to compounds with similar molecular masses.
Trang 8natriuretic 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
10 20
Glial cells
LPS PAO1
LPS PAO9002
0
15
5
1
0.2
0.1
0
EOF
Retention time (min)
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 differdiffer-ent; EOF,
electro-osmotic flux Arrows and numbers
refer to the different molecular forms
identi-fied in the LPS from P aeruginosa
PAO9002.
PAO9002
-PAO9002 BNP
treated
PAO9002 CNP
treated
-+
-+
-+
0
30
40
50
20
10
Fig 8 Cytotoxic activity of P aeruginosa PAO9002 treated with
natriuretic peptides Effect of BNP and CNP (10)6M ) 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.
Trang 9increase 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 propro-tein 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
Trang 10physiological 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
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
collection The vfr null mutant of P aeruginosa PAO1,
(University of Wisconsin-Madison, WI, USA) Both strains
were grown in BNO (Merck, Darmstadt, Germany) at
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
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
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
Glial cell culture
Newborn rats were decapitated 48–72 h after birth, in ster-ile conditions The brain was quickly extracted and rinsed
med-ium (2 : 1) supplemented with 10% fetal bovine serum,
2 mm glutamine, 0.001% insulin, 5 mm Hepes, 0.3%
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
allowed to grow for 9–14 days, to obtain a confluent cul-ture in all culcul-ture wells The culcul-ture 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
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:
ðD 100% 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